Method of generating branched and multi-chain nucleic acid switches for ligand detection

ABSTRACT

Embodiments of the invention relate to a branched or multichain nucleic acid switch adapted to switch from a first conformation to a second conformation upon ligand binding. The switch includes a probe strand, P, which includes the ligand binding domain; a switching framework which includes a cover strand (C), and a tether that holds P and C together and a signaling apparatus. Some embodiments include a toggle strand (T) where now the tether holds P, C, T, and the signaling apparatus together. As the switch changes between the first and second conformations; the signaling apparatus reports the state of the switch. The signaling entity is typically a lumiphore and a quencher located along the switching framework. Nucleic acid switches have applications in real time assays for diverse agents including infectious agents, environmental toxins, and terrorist agents, as well as screening methods for such agents. Further applications are found for nanoelectronics, nanofabrication and nanomachines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/195,547, filed Aug. 2, 2005, now U.S. Pat. No. 7,521,546, whichclaims priority to U.S. Provisional application No. 60/598,498, filedAug. 3, 2004. Both applications are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This work was supported in part by NIH grant R42GM68413 to OrthoSystems,Inc. which has a subcontract to Syracuse University. Consequently, theU.S. government may have certain rights to this invention.

REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING

A sequence listing is annexed hereto.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The disclosed embodiments relate to nucleic acid constructs and methodsof using these constructs to rapidly detect target moleculeinteractions. The switches can have applications in sensing the presenceof targets, including proteins, nucleic acids, organisms, environmentalcontaminants, bioterror agents, and/or pharmaceutical agents. Theswitches should facilitate processes to screen lead compounds for drugdevelopment that have high affinity for a target molecule and/or targetcomplex. Embodiments of the invention to screen potential drugseffective against HIV-1 are used to illustrate the invention. Themolecular switches are also applicable to processes in molecularelectronics and nano-devices.

2. Description of the Related Art

HIV-disease causes great suffering and death in the U.S., and millionsare dying worldwide. Even though the number of deaths in the UnitedStates from HIV-disease has declined in recent years, the worldwideepidemic is out of control. This ever-larger number of infected peopleis a direct threat to everyone because HIV-1 mutates so rapidly. Thelarger the pool of infected individuals, the more rapidly drug-resistantstrains will emerge. The reverse transcriptase makes so many errors thatevery single point mutation occurs daily in newly infected cells(Coffin, J. M. (1995) Science 267:483-9), and nearly 1% of all possibledouble mutations occur (Perelson, A. S. et al. (1997) AIDS 11 (suppl. A)S17-34). Combinations of drugs used in “Highly Active AntiRetroviralTherapy” (HAART) treatment regimes target different parts of the virallife cycle. In the face of such a high mutation rate, it is clear thatfailures in the HAART approach must occur with increasing frequencyusing existing drugs. Resistant strains already exist for all currentlyused protease and reverse transcriptase inhibitors (Pillay, D. et al.(2000) Rev. Med. Virol. 10:231-53), the most potent weapons in thebattle against AIDS.

Even if an effective vaccine is developed to prevent new HIV-1infections, there will still remain a need to treat millions of AIDSvictims. Their long-term treatment will require new generations ofdrugs. Anti-nucleocapsid protein drugs, as well as agents directed atother potential HIV targets, such as anti-rev and anti-tat, could becombined with current and next generation drugs for a multi-prongedattack that would be difficult for the virus to evade. Adding thesedrugs to present HAART treatments may provide highly specific and potentantiretroviral treatments. Such drugs may greatly diminish thedevastating effects of HIV-related disease around the world.

Scores of other human and animal disease states have been related tointeractions of biomolecules with other molecules. These biomoleculespresent targets for therapeutic intervention. This is a current focusfor many academic and industrial efforts to generate newpharmaceuticals. The current invention can accelerate drug discovery.Examples include, but are not limited to, kinases and phosphatasesinvolved in signaling cascades.

Detection of environmental contaminants and terrorist agents has becomean important focus of public concern. The current invention can beapplied to the detection of most agents that interest the EnvironmentalProtection Agency (EPA) (http://www.epa.gov/safewater/mcl.html#mcls) andon the select agents list monitored by the Centers for Disease Control(CDC), the National Institute of Allergy and Infectious Diseases (NIAID)(http://www2.niaid.nih.gov/Biodefense/bandc_priority.htm), and theHomeland Security Agency (HSA). The current invention provides atechnology for near real-time detection of environmental and terroristagents. Examples include, but are not limited to, cryptosporidium andgiardia, which contaminate public water supplies, and ricin, anthrax andebola virus, which are agents for bio-terrorism and bio-warfare.

Molecules that can switch between stable states are of interest fornanoelectronics, nanofabrication, and nanomachines. The presentinvention is of special interest in high density information storagedevices used in nanoelectronic applications. Read, write, and erasefunctions can be constructed using the subject invention. It is alsocontemplated that subject molecular switches could be coupled to buildmaterials and machines on the molecular scale.

SUMMARY OF THE INVENTION

Embodiments of the invention are directed to a branched or multichainnucleic acid switch adapted to switch from a first conformation to asecond conformation upon ligand binding. The switch may include one ormore of the following elements:

-   -   a probe strand P which includes at least one ligand binding        domain;    -   a switching framework which includes a cover strand (C);    -   a tether that holds P and C together while the switch changes        between the first and second conformations; and    -   a signaling apparatus which includes a combination of signaling        entities.

Preferably, the probe strand is DNA, RNA, modified nucleic acid, orcombinations thereof. In preferred embodiments, the first conformationhas P extensively hybridized to C and the second conformation has P nothybridized to C. Preferably, C is at least 50% complementary to P. Morepreferably, C is completely complementary to P.

In preferred embodiments, the signaling entities include a lumiphore anda quencher located along the switching framework.

In preferred embodiments, the branched or multichain switch alsoincludes a toggle strand (T). Preferably, T is at least 50%complementary to P.

In preferred embodiments, the branched or multichain switch alsoincludes a fastener, F, to couple two nucleic acid strands together.Preferably, the first strand includes C and the second strand includes Pwhen the branched or multichain switch is in the second conformation.

In preferred embodiments, the ligand binding domain of the branched ormultichain switch includes a naturally occurring RNA binding site oranalog thereof or a naturally occurring DNA binding site or analogthereof or a combinatorially derived sequence or related fragment.

Preferably, the ligand binding domain of the branched or multichainswitch is adapted to bind a ligand which is selected from:

-   -   a disease agent, such as an agent from a disease including but        not limited to Hepatitis C, Congo-Crimean hemorrhagic fever,        Ebola hemorrhagic fever, Herpes, human cytomegalovirus, human        pappiloma virus, influenza, Marburg, Q fever, Rift valley fever,        Smallpox, Venezuelan equine encephalitis, HIV-1, MMTV, HIV-2,        HTLV-1, SNV, BIV, BLV, EIAV, FIV, MMPV, Mo-MLV, Mo-MSV, M-PMV,        RSV, SIV, or AMV;    -   a retroviral component including but not limited to TAR-tat,        RRE-rev, DIS, PBS, RT, PR, IN, SU, TM, vpu, vif, vpr, nef, mos,        tax, rex, sag, v-src, v-myc and precursors and protease products        of the precursors, gag, gag-pol, env, src, or one;    -   a toxin or other factor derived from bacteria or other        microorganisms including but not limited to B. anthracis,        Burkholderia pseudomallei, Botulinum, Brucellosis, Candida        albicans, Cholera, Clostridium perfringins, Kinetoplasts,        Malaria, Mycobacteria, Plague, Pneumocystis, Schistosomal        parasites, Cryptosporidium, Giardia, Ricin, Saxitoxin, Shiga        Toxin, Staphylococcus (including enterotoxin B), Trichothecene        mycotoxins, Tularemia, or agents causing Toxoplasmosis; and    -   nerve gas agents, chemical poisons, contaminants of water        supplies, contaminants of food and beverages, or contaminants of        air.

Embodiments of the invention are directed to populations of branched ormultichain switch molecules which include P and C and optionally T.Preferably, there are more molecules with C hybridized to P than with Pnot hybridized to C. In other preferred embodiments, there are moremolecules with C hybridized to P than with T hybridized to C.

In preferred embodiments of the branched or multichain switch, P, C andT are joined together at a vertex.

Embodiments of the invention are directed to methods to generate thebranched or multichain switches described above by adjusting theequilibrium constant, K1, for the switch from the first conformation tothe second conformation. In preferred embodiments, the sequence of C isaltered, and the free energy difference between the two conformations,is used to estimate K1. In preferred embodiments, K1 is set to favorconformation 1. Preferably, the value of K1 can be experimentallyverified. In preferred embodiments, a signal of a candidate switch isused to determine the candidate's value of K1 by interpolation betweenthat for sequence (a), which favors conformation 1 over conformation 2by a factor of 100 or more, and sequence (b), which favors conformation2 over conformation 1 by a factor of 100 or more. In preferredembodiments, the value of K1 for switch candidates is estimated bycomparison with known competitors, X, of the ligand, L, designed tointeract with conformation 2 of the switch.

Embodiments of the invention are directed to methods to generate thebranched or multichain switches which include P, C, and T which includeat least one of the following steps:

-   -   adjusting an equilibrium constant, K1, for the switch from the        first conformation to the second conformation;    -   altering sequences of C and T; and    -   estimating K1, based upon the free energy difference between the        two conformations.

Embodiments of the invention are directed to a kit which includes thebranched or multichain switch including P, C, and optionally, T for realtime detection of a selected ligand.

Embodiments of the invention are directed to methods of chemicalscreening which include one or more of the following steps:

-   -   providing a bistable branched or multichain switch, which        includes an analog of an RNA or DNA, wherein the analog includes        the ligand binding domain, a molecular framework and a signaling        apparatus;    -   contacting the branched or multichain switch with the ligand in        the absence of a screened chemical entity;    -   monitoring a signal produced from the signaling apparatus in the        absence of the chemical entity;    -   contacting the branched or multichain switch with the ligand in        the presence of the chemical entity;    -   monitoring a signal produced from the signaling apparatus in the        presence of the chemical entity; and    -   comparing the signal produced in the absence of the chemical        entity with the signal produced in the presence of the chemical        entity to determine the effect of the chemical entity on the        ligand binding.

In preferred embodiments, the signaling apparatus includes a lumiphoreand a quencher of the lumiphore. In preferred embodiments, the ligandbinding domain includes RNA, the molecular framework includes DNA andthe ligand is a viral protein.

In preferred embodiments, the ligand binding domain includes RNA, themolecular framework includes DNA and the ligand binding domain includesa naturally occurring RNA binding site or analog thereof or a naturallyoccurring DNA binding site or analog thereof or a combinatoriallyderived sequence or related fragment. Preferred embodiments also includethe step of equilibrating the molecular switch and the ligand prior toadding the chemical entity.

In preferred embodiments, the ligand binding domain includes RNA, themolecular framework includes DNA and the ligand is selected from:

-   -   a disease agent wherein the disease is Hepatitis C,        Congo-Crimean hemorrhagic fever, Ebola hemorrhagic fever,        Herpes, human cytomegalovirus, human pappiloma virus, influenza,        Marburg, Q fever, Rift valley fever, Smallpox, Venezuelan equine        encephalitis, HIV-1, MMTV, HIV-2, HTLV-1, SNV, BIV, BLV, EIAV,        FIV, MMPV, Mo-MLV, Mo-MSV, M-PMV, RSV, SIV, or AMV;    -   a retroviral component which is TAR-tat, RRE-rev, DIS, PBS, RT,        PR, IN, SU, TM, vpu, vif, vpr, nef, mos, tax, rex, sag, v-src,        v-myc and precursors and protease products of the precursors,        gag, gag-pol, env, src, or one;    -   a toxin or other factor derived from bacteria or other        microorganisms which are B. anthracis, Burkholderia        pseudomallei, Botulinum, Brucellosis, Candida albicans, Cholera,        Clostridium perfringins, Kinetoplasts, Malaria, Mycobacteria,        Plague, Pneumocystis, Schistosomal parasites, Cryptosporidium,        Giardia, Ricin, Saxitoxin, Shiga Toxin, Staphylococcus        (including enterotoxin B), Trichothecene mycotoxins, Tularemia,        or agents causing Toxoplasmosis; and    -   nerve gas agents, chemical poisons, contaminants of water        supplies, contaminants of food and beverages, or contaminants of        air.

Embodiments of the invention are directed to a molecular switch adaptedto switch from a first energy state to a second energy state uponapplication of triggering photons, said switch including one or more ofthe following:

-   -   a photosensitive ligand binding domain which includes a        combinatorially-derived sequence;    -   a molecular framework which includes the photosensitive ligand        binding domain adapted to switch from a first stable        conformation to a second stable conformation upon binding of the        photosensitive ligand by the ligand binding domain; and    -   a signaling apparatus along the molecular framework which        includes a combination of signaling entities that vary upon        application of the triggering photons when the molecular        framework switches between first and second stable        conformations.

Embodiments of the invention are directed to methods of using any of themolecular switches described above, including at least the steps of:

-   -   binding a ligand to the ligand binding domain in P, whereby the        branched or multichain switch changes from the first        conformation to the second conformation upon ligand binding; and    -   measuring a luminescence change as a result of the        conformational change.

Embodiments of the invention are directed to a branched or multichainswitch adapted to switch from a first conformation to a secondconformation upon ligand binding, said switch including a first strandwhich has a ligand binding domain; a second strand; and a fastener tocouple the two strands together; wherein the first strand and secondstrand hybridize to each other in the first conformation and the ligandbinding domain is free in the second conformation.

Further aspects, features and advantages of this invention will becomeapparent from the detailed description of the preferred embodimentswhich follow.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other feature of this invention will now be described withreference to the drawings of preferred embodiments which are intended toillustrate and not to limit the invention.

FIG. 1 is a schematic view of packaging in HIV-1 as a new virus particleassembles at the membrane of an infected cell. Each virus has twoidentical RNA strands, about 2000 gag and gag-pro-pol precursorproteins, and several other proteins and cellular components. As thevirus matures, the precursors are processed into separate proteins,including NCp7. Within the precursor, the NC domains recognize sets oftwo loop G-residues (G₂ loci) in the RNA 5′-leader.

FIG. 2 shows the HIV-1 NCp7 protein (SEQ ID NO: 8). Residues carrying acharge at neutral pH are shown in outlined letters (positive=capitals,negative=lower-case); Zn₂.NCp7(1-55) carries a +9 charge at neutral pH.

FIG. 3 is a hypothetical secondary structure of the HIV-1 RNA domainnear the 5′-major splice donor (SD) (SEQ ID NO: 9) (Pappalardo, L. etal. (1998) J. Mol. Biol. 282:801-818). Several known and potentialG₂-loci are noted.

FIG. 4 is a detailed view of part of the packaging signal interaction,showing the SL3-NCp7 complex (DeGuzman, R. et al. (1998) Science279:384-388). It can be seen that W37 of the protein stacks on G318.

FIG. 5 illustrates quenching W37 in NCp7 by four HIV-RNA molecules. ♦SL3-UUCG, ● SL4, ▴ SL3, ▪ 154mer full domain; solid lines representcalculated fits for 1:1 complexes (see text). Short dashed line: idealR₁L₃ complex, long dashes: ideal R₁L₃ complex. R_(t), and L_(t) are thetotal concentrations of RNA and ligand (NCp7), respectively.

FIG. 6. (a) Equilibria for complex formation between a protein, P,competitor, C, and a nucleic acid switch. (b) The “C3” sequence (SEQ IDNO: 7) used in phase I is shown, with the H-form at the left and O-format the right. DNA is shown in red and RNA in blue, gray blocks designatebase pairs in O that are lost in H, the high affinity GUG sequence is inthe blue oval, * is a fluorescein derivative, 6-hydroxy fluorescein(FAM), and Q is the fluorescence quencher, Dabsyl that replaces the5-methyl of T.

FIG. 7. Fluorescence of the C3 (SEQ ID NO: 7) (heavy blue line, secondfrom the top, at concentration, N_(t)=10 nM). Additions of NCp7 areshown in light lines (21 to 575 molar equivalents of protein). The heavyred line (bottom) is for the P_(t)/N_(t)=575 sample treated with RNase,and the heavy green line (top) is for that sample treated with DNase.

FIG. 8. FRET-monitored titration of the C3 (SEQ ID NO: 7) (N_(t)≅10 nM).[NCp7] increases left to right, expressed as the ratio P_(t)/N_(t). Theemission at 517 nM is reported after subtracting that of the RNasetreated sample. The fit uses a single binding constant K_(d)′=500 Nm.Uncertainty in N_(t) limits the precision of this determination.

FIG. 9. Increase in fluorescence upon addition of SL2 to a quenchedC3/NCp7 complex. [C3]≅10 nM.

FIG. 10. Increase in fluorescence upon addition of SL2 to a quenchedC3/NCp7 complex. [C3]≅10 nM.

FIG. 11 shows the equilibria for the SL3-NCp7 assay using a tetheredluminescent switch in accordance with the first embodiment of thepresent invention. The outline of a luminescence assay used to detectcompetitors of SL3-NCp7 complex formation is shown in the inset (topright). The sequences denote a tethered switch with two stable states:one where the binding site (GGUG sequence) is hidden, H, at left, andone where the binding site is open, O, at right. RNA segments aredenoted in dark italics, DNA in lighter font. Tethering is accomplishedby the fifteen base-pair stem that does not vary between the two statesof the switch. The figure shows M3 (SEQ ID NO: 3) and D2 (SEQ ID NO: 6)as the two strands.

FIG. 12 shows competitor thresholds for the luminescent RNA/DNA chimeraillustrated in FIG. 11, total concentration of switch molecules,O_(t)=0.3 μM, total concentration of ligand molecules, L_(t)=3 μM,K₁=0.1 nM, K₂=3 nM. Values of K₃ are indicated below the graph. SeeDetailed Description of the Preferred Embodiment for definitions ofequilibrium constants, K₁, K₂, and K₃.

FIG. 13 illustrates the embodiment in which probe (P), cover (C), andtoggle (T) strands are tethered at a single vertex, thus comprising aunimolecular switch.

(a) A 3-fold junction connects the P, C, T segments, and the designationT_C_P denotes the unpaired random-coil reference state for free energycomparisons.

(b) The bistable molecule can exist in either the T_C:P state or theT:C_P state. The ligand binding site in P is hidden (sequestered in abase-paired region) in T_C:P and open in T:C_P. The distances betweenthe signaling entities, S, S′, and S″, change when the switch changesstate.

(c) A free energy diagram depicts interconversion of states. The twoforms of the switch convert rapidly if the intermediate steps occur viabranch migration.

(d) The sequences of the three segments can be engineered to create anefficient molecular switch. A “1” denotes a nucleotide complementary toa “2”, and “3” is complementary to “4”. The “combimer” contains theligand binding site, in P, which is written 5′-3′, left to right. Thecover segment, C, written 3′-5′, can be slightly longer than P, butotherwise is depicted as fully complementary. The toggle segment, T, isdepicted as having a mismatch with C in the combimer zone, but isotherwise fully complementary.

FIG. 14 illustrates three embodiments which are analogous to FIG. 13 b.The fastener stem, F, is stable and does not substantially alter duringswitching events. P, C, and T have the same meaning as in FIG. 13.Locations of signaling moieties, S, S′, and S″ can be optimized tocreate the most robust signal output.

FIG. 15 illustrates an embodiment in which P and C lie on separatestrands, which are held together by stable fastener stems, F1 and F2.

FIG. 16 shows a basic module for molecular electronic applications. Top:A preferred digital system has states “Zero” and “One”, which areswitched by application of light at frequencies ν₁ (Write) and ν₂(Erase); the state of the system is interrogated at ν₃ (Read) anddetected at ν₄. Bottom: Schematic of switchable nucleic acid constructsto support the preferred embodiment. S denotes a site for possibleattachment to a solid support.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

FRET stands for fluorescence resonance energy transfer,

The term, luminescence, is a broad term and is used to encompass allforms of the emission of light subsequent to excitation by light. Thisincludes fluorescence as it is commonly encountered in FRET andmolecular beacon technology where emission or quenching occurs withinmicroseconds of excitation. It also encompasses longer-livedphosphorescence, and emission from lanthanides where spin-orbit couplingis so large that fluorescence and phosphorescence cannot bedistinguished. Reference will often be made to molecular beacontechnology using conventional fluorophores and quenchers. However, it isunderstood that the signaling apparatus is not limited to conventionalfluorophores and quenchers; substitution of another excitation-emissionscheme, such as with sensitized lanthanides and appropriate quenchingmoieties is also included within the scope of this invention.

A lumiphore is a chemical entity that produces luminescence,particularly a chemical entity that that absorbs electromagnetic energy,then emits energy by fluorescence, phosphorescence, or luminescence.

A refers to a bistable RNA and/or DNA construct (except where A is usedto designate adenine in a nucleotide sequence),

X refers to any molecule that competes with or otherwise interferes withbinding of L to natural RNA or DNA analogs of A,

L, L1, L2, etc. refer to a ligand molecules such as a protein,cell-surface feature, small molecule, or other chemical entity thatbinds to the O-form of a molecular switch,

O refers to the open conformation of a switch molecule where the bindingsite is available to interact with the ligand,

H refers to the switch conformation where the binding site is hidden,

K₁=[O]/[H] refers to the H⇄O equilibrium constant, where [x]denotes theconcentration of species, x,

K₂=[L][O]/[L.O] refers to the L.O⇄L+O dissociation equilibrium constant,

K₃=[L][X]/[L.X] refers to the L.X⇄L+X dissociation equilibrium constant,

Q refers to a quencher in a conventional FRET or molecular beaconexperiment, or where longer-lived luminescence is quenched,

*, D, D1, D2, etc. refer to a fluorescence donor in a FRET or molecularbeacon experiment, or longer-lived excited states in luminescent donors,

S, S′, S″, etc. refer to signaling moieties that include FRET donors andacceptors, luminescent constructs, etc.,

F, F1, F2, etc. refer to fastener duplex sequence(s) required fortethering separate strands of a molecular switch. These fixed sequencesare not varied to optimize the performance of a switch,

P refers to a probe segment that contains a ligand binding site in the Oconformation of a molecular switch,

C refers to a cover segment that hides the probe sequence in the Hconformation of a switch,

T refers to a toggle segment that is present in certain molecularswitches.

Embodiments of the invention relates to the design and application ofbistable RNA and/or DNA constructs (A) that are switchable between twothermodynamically stable states. In preferred embodiments, theconstructs are composed partially or entirely of mimetics of nucleicacids. One of the stable states includes a site for binding a targetprotein, nucleic acid, saccharide, small molecule, or supramolecularassembly. This binding site is sequestered in the second stable state.The detailed nature of the construct depends on the target. An exampleis provided for discovery of small molecules that inhibit the formationof a natural RNA-protein complex. A molecular switch with the desiredproperties is illustrated in a construct that tethers an all-DNA strandto an RNA-DNA chimeric strand. Other targets may bind natural RNA or DNAsequences, or the binding elements of the switch may be discovered viain vitro experiments to choose “combinatorially-derived sequence”molecules. The technology disclosed herein enables one skilled innucleic acid chemistry and biophysical chemistry to design a suitablebistable construct and then to fine-tune the relative stability of thetwo forms.

Areas of Contemplated Use

(1) Embodiments of the invention are directed to diagnostic tests forthe presence of a protein, nucleic acid, supramolecular structure, wholeor inactivated organism, or other ligand molecule (L) that bindspreferentially to one of the two stable states of A. This stable statecontains an analog of a naturally occurring RNA or DNA binding site forL (ligand binding domain).

(2) Embodiments of the invention are directed to the discovery ofchemical entities (X) that interfere with binding of L to natural RNA orDNA analogs of A. One application involves X molecules that are leadsfor therapeutic agents against a disease state for which A-Linteractions are necessary, e.g., interactions between SL3 of HIV-1 RNAand NCp7.

(3) Embodiments of the invention are directed to applications similar to(1), wherein the ligand binding domain of A comprises acombinatorially-derived sequence that is empirically chosen to bindtightly and specifically to L. Embodiments include field kits forreal-time detection of infectious organisms or toxic agents.

(4) Embodiments of the invention are directed to applications similar to(2), wherein the ligand binding domain of A comprises acombinatorially-derived sequence that is empirically chosen to bindtightly and specifically to L. Embodiments include the discovery ofchemical agents, X, for the remediation of effects due to infectious ortoxic agents, L.

(5) Embodiments of the invention are directed to molecular electronicapplications where the state change in A occurs in response to atriggering impulse, which may be a light pulse, that alters the state ofa photosensitive ligand, L1, to L2. In these applications, the ligandbinding domain of A may contain a natural RNA or DNA binding site for L1or L2, or a combinatorially-derived sequence empirically chosen to bindtightly and specifically to either L1 or L2. The shape and properties ofA will depend upon whether the combinatorially-derived sequence-bindingpocket is occupied. Here, the construct may include a lumiphore quencherpair or other signal generating elements.

Embodiments of the invention rely on a conformationally bistableconstruction for A that is switched from one state (A1) to the other(A2) upon binding L. In the A2-L complex, the molecular conformation,A2, differs from that which predominates in the unbound state, A1. Thestate change may be detected by a change in luminescence, because theluminescent properties of A1 and A2 are designed to be very different.The fraction of A that is present as the A2-L complex is controlled byseveral thermodynamic factors, including (i) the relative affinity of Lfor A1 and A2, (ii) the A1/A2 equilibrium, and (iii) the inputconcentrations of the species. Some applications will includecompetitors, X, for the A2-L interaction, in which case the affinitiesof X for L, A1, and A2 are also relevant.

An embodiment of item (2), above, is used to illustrate the invention,where a bistable RNA/DNA construct including a lumiphore-quencher pairis disclosed for the rapid screening of agents to disrupt the SL3-NCp7interaction in HIV-1. That embodiment is described in detail below. Inthat description, H, and O, replace the general nomenclature, A1 and A2,respectively.

Certain embodiments of the invention require the construction of an RNAand/or DNA (or other natural or nucleotide mimetic analogs) moleculethat is switchable between two thermodynamically stable states.Illustrations and working examples are disclosed for double-hairpinconstructs and cruciform structures.

HIV-1 has fifteen proteins and two identical RNA strands. Each of theseis a potential target for drug interdiction. More details are given inseveral reviews and books (Frankel, A. D. and Young, J. A. T (1998) Ann.Rev. Biochem. 67:1-25; Coffin, J. M. et al. (1997) Retroviruses, ColdSpring Harbor Lab. Press, Plainview, N.Y.; Gallo, R. C., & Jay, G., eds.(1991) The Human Retroviruses, Academic Press, New York). Drugscurrently in use target the viral reverse transcriptase (RT) andprotease (PR). There are efforts to develop inhibitors of other HIVproteins. Several other HIV targets have been suggested. These includethe nucleocapsid protein, as well as tat and rev (Frankel, A. D., andYoung, J. A. T (1998) Ann. Rev. Biochem. 67:1-25).

For HIV to reproduce, genomic RNA and viral proteins must form a“packaging complex,” illustrated in FIG. 1. The 5′-leader region of theRNA contains sequences that allow only infectious RNA to be packagedinto new virus particles, selected from the millions of other RNAmolecules in a cell.

The NC domain or mature NCp7 has roles in packaging the RNA (Wills, J.W., & Craven, R. C. (1991) AIDS 5:639-654; Oertle, S, and Spahr, P.(1990) J. Virol. 64:5757-5763; Damgaard, C. K. et al. (1998) NucleicAcids Res. 26:3667-76; DeGuzman, R. et al. (1998) Science 279:384-388),“chaperoning” functions (Williams, M. C. et al. (2001) Proc Natl AcadSci USA 8:8), in refolding the RNA dimer in the virion (Fu, W. and Rein,A. (1994) J. Virol. 68:5013-5018), and annealing the primer tRNA ontothe genomic RNA for reverse transcription (Prats, A. C. et al. (1988)EMBO J. 7:1777-1783). It also interacts with viral proteins includingreverse transcriptase (Druillennec, S. et al. (1999) J. Biol. Chem.274:11283-8; Lener, D. et al. (1998) J. Biol. Chem. 273:33781-6), andthe accessory protein, Vpr, to play a role in stable integration of theproviral DNA in the chromosomes of infected cells (de Rocquigny, H. etal. (2000) Eur. J. Biochem. 267:3654-60). Thus, drugs that target thenucleocapsid protein and/or its interactions with other HIV-1 moleculeshave the potential to interfere with critical functions at many stagesof the viral life cycle (Darlix, J. L. et al. (2000) Adv. Pharmacol.48:345-72; Berthoux L. et al. (1999) J. Virol 73:10000-9).

The NC, tat, and rev proteins all interact with RNA in regions that canbe created with the luminescent RNA/DNA chimeras in accordance withpreferred embodiments of the present invention, as described below ingreater detail. Thus, those of ordinary skill in the art will appreciatethat the compositions and methods used to screen anti-NC candidatesdisclosed herein can be readily adapted for these other HIV-1 targets,as well as molecular targets in other disease states, or to detect thepresence of a protein, organism, toxin, or other target.

Anti-NC Strategies

A goal of targeting an enzyme with an equilibrium-binding agent is todecrease the enzyme's turnover rate. Small changes in the binding freeenergy of competitors may be amplified by exponential decreases inturnover. Thus, drugs having micromolar or even millimolar affinitiesmay be sufficiently effective as long as Absorption, Distribution,Metabolism, Elimination, Toxicity (“ADME/Tox”) properties are favorable.This is the fundamental advantage in targeting RT, PR, or IN over viralsubstances that exert their influence by mass action. On the other hand,this amplifier effect confers a strong survival advantage on mutantsresistant to a drug. In a short time, a mutant strain can dominate aninfection.

The mature NCp7 protein presumably turns over in its chaperoningactivity. Thus, it is possible there will be an amplifier effect similarto that for equilibrium binding drugs to inhibit RT, PR, or IN. Thepackaging function of NC, however, seems to be dominated by massaction—2000 gag precursor proteins are available to recognize thepackaging domain RNA for each virus. An answer to mass action is tighterbinding. Thus, the preferred target drugs will exhibit high affinitybinding to the HIV-1 RNA.

A possible way to skirt problems due to low affinity or a mass-actionfunction is for a drug to covalently inactivate the target. There havebeen attempts to adapt sulfur-reactive compounds to attack cysteine sidechains in the zinc fingers (Rice W G et al. (1993) Nature 361:473-5;Rice W G et al. (1995) Science 270:1194-7; Chertova E N et al. (1998)Biochemistry 37:17890-7; Huang M et al. (1998) J Med Chem. 41:1371-81;Guo J et al. (2002) J Virol. 76:4370-8; Yovandich J L et al. (2001) JVirol. 75:115-24; Berthoux, L et al. (1999) J Virol 73:10000-9). Cys49,in the C-terminal finger (FIG. 2), reacts fastest with N-ethylmaleimide(NEM) in vitro (Chertova E N et al. (1998) Biochemistry 37:17890-7). Acascade of reactions then ejects Zn²⁺ from both fingers. The reactionwith NEM is slow even at relatively high concentrations (˜8 mM NCp7, 50mM NEM; t_(1/2)≅30 min for forming the C49 adduct).

Other “zinc-ejecting” alkylating agents have been tested (Rice W G etal. (1993) Nature 361:473-5; Rice W G et al. (1995) Science 270:1194-7;Chertova E N et al. (1998) Biochemistry 37:17890-7; Huang M et al.(1998) J Med Chem. 41:1371-81; Guo J et al. (2002) J Virol. 76:4370-8;Yovandich J L et al. (2001) J Virol. 75:115-24; Berthoux, L et al.(1999) J Virol 73:10000-9). However, cysteines are common, as arezinc-chelating proteins. Thus, zinc ejection seems to violate theprinciple that one should attack HIV at processes that are specific tothe virus.

In an attempt to counter this concern, some reagents were tested withzinc-containing transcription factors. The experimenters suggested thatthe reagents were selective for reaction with NC, while the cysteineschelating zinc in the fingers of the transcription factors were notalkylated. However, examination of the protocol (Huang M et al. (1998) JMed Chem. 41:1371-81) shows that the latter reaction was conducted inthe presence of the DNA substrate for the cellular protein. Alkylationof Cys49 can be stopped almost completely by preincubating NCp7 withd(GT)_(n) oligomers in 2-fold excess (Chertova E N et al. (1998)Biochemistry 37:17890-7); these bind much more weakly than the naturalRNA substrates for NC (see EXAMPLES). Selectivity then remains an openquestion. It is likely that both NC and cellular transcription factorsare present mostly as RNA or DNA-bound forms.

Others have experimented with short single-stranded DNA molecules(Vuilleumier, C et al. (1999) Biochemistry 38:16816-25; Mely, Y et al.(1994) Biochemistry 33:12085-91; Maki, A. H. et al. (2001) Biochemistry40:1403-1412; Fisher, R. J. et al. (1998) J Virol 72:1902-9), or DNA andNC mimetics (Druillennec S et al. (1999) Bioorg Med Chem Lett. 9:627-32;Druillennec S et al. (1999) Proc Natl Acad Sci USA. 96:4886-91). TheK_(d) values are probably in the millimolar range at best, although thisis difficult to confirm.

In work on deoxy dinucleotides where the phosphodiester was replaced bya methylene carboxamide linker (Druillennec S et al. (1999) Bioorg MedChem Lett. 9:627-32), K_(d) values for NC binding were estimated at 6 μMfor TG, 100 μM for GT, and >1000 μM for TT. Protein binding for thedinucleotides is considerably weaker than for SL3 or SL2 RNA, but it isencouraging that uncharged molecules with MW˜500 exhibit both affinityand sequence specificity. The other conclusions are that the TGmolecule: (i) penetrates cells in an HIV-1 infected cell line, (ii)inhibits RT activity by 20% at a concentration of 10 μM, and (iii)appears to snap NC into the same binding conformation given that shiftsin the NMR spectrum of NC are similar to those induced by longer nucleicacids.

A cyclic peptide that competes with NC for in vitro recognition of itsRNA, DNA and protein targets may function as a mimic for NC(Druillennec, S et al. (1999) Proc Natl Acad Sci USA 96:4886-91). Thishexapeptide, c(F-C-dW-R-C-K), has strong structural similarities in thelocations of W, F, and basic side chains in NCp7. It also exhibits Invivo effects that suggest impairment of proviral DNA synthesis, perhapsby direct interaction with RT or by interfering with annealing the tRNAprimer to genomic RNA.

There is also some enthusiasm for competitive inhibitors based on RNAaptamers that have been created with nanomolar affinities for NCp7(Lochrie, M. A. et al. (1997) Nucleic Acids Res 25:2902-10; Berglund, J.A. et al. (1997) Nucleic Acids Res 25:1042-9; Allen, P. et al. (1996)Virology 225:306-15). Most of the binding studies were conducted at lowsalt and the stoichiometry was not clearly established. Therefore, someof these molecules are considered as possible candidates herein.However, unmodified DNA and RNA molecules may not readily pass the cellmembrane and survive long enough in a cell to disrupt the target NC-RNAinteraction.

The present screening methods are applicable for the high-throughputassay of low-molecular weight lead compounds. Generally, low-MW drugsare more permeable to cell membranes than macromolecules, are accessibleby organic synthesis, and pharmaceutical companies are experienced informulating similar compounds for oral dosage. Libraries of thousands of“drug-like” compounds are now available for high throughput screening.They possess diverse molecular scaffolds to locate lead compounds, whichcan be modified by combinatorial and rational design to optimize theirNC affinity, and ADME/Tox properties.

The compounds and methods disclosed with respect to the NC-RNA systemshould be directly applicable for developing and testing useful low-MWcompounds. Indeed, the work in measuring affinities, in structuredetermination by NMR, and stabilizing NCp7 against denaturation,disclosed herein, can be applied in a vigorous program ofanti-nucleocapsid drug discovery and design.

Structural Biology of Packaging

FIG. 3 shows a plausible secondary folding of the major packaging domainof HIV-1 RNA. Several elements for controlling the viral life cycle arecontained within this ˜150 nucleotide sequence. (1) A metastable RNAdimer forms around the dimer initiation sequence (DIS) (Muriaux, D. etal. (1996) Biochemistry 35:5075-82; Muriaux, D. et al. (1996) J BiolChem 271:33686-92; Clever, J. L. et al. (1996) J Virol. 70:5902-8;Laughrea, M. et al. (2001) Virology 281:109-116) in SL1, which thenmatures in the virion to a more stable form condensed with NCp7 (Fu, W.& Rein, A. (1994) J Virol. 68:5013-5018). The mature virus has about oneNCp7 per 10-12 nucleotides. (2) The 5′-major splice donor (SD) in SL2 isa primary RNA processing site. Since spliced mRNA is not packaged, it islikely that packaging determinants reside in the sequence or foldedstructure of the region near SD. (3) A determinant of packaging is SL3,in which two nearby guanine residues (G₂-loci) appear to be involved inspecificity; G₂-loci in other stem-loops are also involved. Thefollowing paragraphs give a more complete description of the backgroundto packaging. (4) The coding region for the gag genes begins in SL4.

Many details of packaging in retroviruses have come into focus (Coffin,J. M. et al. (1997) Retroviruses, Cold Spring Harbor Lab. Press,Plainview, N.Y.; Gallo, R. C., & Jay, G., eds. (1991) The HumanRetroviruses, Academic Press, New York; Clever, J. L. et al. (1999) JVirol. 73:101-9; Clever, J. L. & Parslow, T. G. (1997) J Virol.71:3407-14; McBride, M. S. & Panganiban, A. T. (1996) J Virol.70:2963-73; McBride, M. S. et al. (1997) J Virol. 71:4544-54; Clever, J.et al. (1995) J Virol 69:2101-9). In HIV-1 about 1500-2500 polyproteinprecursors (pr-gag, and pr-gag-pol) assemble at the inner membrane ofthe forming virion (Vogt, V. M. & Simon, M. N. (1999) J Virol 73:7050-5)rather than just the few illustrated in FIG. 1. Each of these proteinscontains a nucleocapsid domain that is required for packaging to occur.The 55 kD gag precursor polyprotein is later processed by the viralprotease to “structural” proteins, including NCp7 (Linial, M., & Miller,A. D. (1990) Curr. Top. Microbiol. Immunol. 157:125-152; Gelderblom, H.R. (1991) AIDS 5:617-638). NC-domains within gag precursors bind to theRNA with several RNA-NC interactions responsible for full discriminationof genomic from non-genomic RNA (Clever, J. L. et al. (1999) J Virol.73:101-9; Clever, J. L. & Parslow, T. G. (1997) J Virol. 71:3407-14;McBride, M. S. & Panganiban, A. T. (1996) J Virol. 70:2963-73; McBride,M. S. et al. (1997) J Virol. 71:4544-54; Clever, J. et al. (1995) JVirol 69:2101-9). The NC-domains interact via a conserved zinc fingermotif (FIG. 2). Mutants of the fingers that render them incompetent forzinc binding destroy the capacity to recognize and package genomic RNA(Aldovini, A. & Young, R. A. (1990) J Virol 64:1920-6; Gorelick, R. J.et al. (1988) Proc Natl Acad Sci USA 85:8420-4; Dupraz, P. et al. (1990)J Virol 64:4978-87). It is thought that interactions with the twofingers in NCp7 are the same as in the precursor.

Most HIV-1 packaging specificity occurs with sequences encompassed bythe nucleotides shown in FIG. 3 (Pappalardo, L. et al. (1998) inStructure, Motion, Interaction and Expression of BiologicalMacromolecules, pp. 125-135 (R. H. Sarma & M. H. Sarma, eds.); Shubsda,M. F. et al. (2002) Biochemistry 41:5276-82). There have also beenmeasurements of the association constants of various RNA fragments withthe 15 kDa NCp7 precursor protein (Clever, J. et al. (1995) J Virol69:2101-9), and GST-NC fusion proteins (McBride, M. S. & Panganiban, A.T. (1996) J Virol. 70:2963-73) indicating that all four hairpins areinvolved with NC-domain interactions. Each loop is a favorable candidatefor interaction with the NCp7 zinc-finger domain, with 2 or 3 G residuesin single-stranded loops; G may be a requirement for interaction withthe finger in the native RNA sequence (South, T. L. & Summers, M. F.(1993) Protein Sci 2:3-19; Summers, M. F. et al. (1992) Protein Sci,1:563-574; Delahunty, M. D. et al. (1992) Biochemistry 31:6461-6469).Removal of SL3 reduces packaging efficiency by ˜90%, but does notcompletely eliminate packaging (Clever, J. L. & Parslow, T. G. (1997) JVirol. 71:3407-14). This suggests that RNA-NC domain interactions mayoccur at several sites to provide full specificity.

A high-resolution view of a packaging signal complex is illustrated inFIG. 4 (DeGuzman, R. et al. (1998) Science 279:384-388; Pappalardo, L.et al. (1998) J. Mol. Biol. 282:801-818). FIG. 4 shows the complexbetween a 20mer SL3 construct and the 55mer NCp7. No NMR or x-raystructures for complete retroviral packaging signals have been reported,although there are several other structures for substantial subsets(Amarasinghe, G. K. et al. (2000) J. Mol. Biol. 299:145-156;Amarasinghe, G. K. et al. (2000) J. Mol. Biol. 301:491-511; Kerwood, D.J. et al. (2001) Biochemistry 40:14518-29; Mujeeb, A. et al. (1998) NatStruct Biol 5:432-436; Takahashi, K. I. et al. (2000) RNA 6:96-102;Theilleux-Delalande, V. et al. (2000) Eur J Biochem 267:2711-2719;Ennifar E et al. (2001) Nat Struct Biol. 8:1064-8; Zeffman, A. et al.(2000) J. Mol Biol. 297:877-93; Morellet, N. et al. (1998) J. Mol. Biol.283:419-34). There is a high degree of similarity of the interactionsbetween NCp7 and nucleic acid in these complexes. Thus it is likely thatthe development and testing of anti-nucleocapsid drugs can be guided bymolecular modeling based on the conserved structure.

FIG. 3 shows that splicing would destroy this secondary structure (SD isat 289-290), removing essential portions of SL2 and sA as well as all ofSL3 and SL4. That provides a natural explanation for the selection ofunspliced RNA for packaging. We have examined about 500 sequences inGenbank containing SL3, and found (Lin, Y. (2002) Ph.D. Thesis, SyracuseUniversity; “Database and Algorithmic Applications in Nucleic AcidSequence, Structure and NMR Frequencies, and in Efficient ChemicalDepiction.”; which is incorporated herein in its entirety by reference)that only the first and third base in the GGAG tetraloop of SL3 varymore than twice (about the rate of sequencing errors). The G₂-locus at318 and 320, which is involved in the specific complex of FIG. 4, may berequired for a functioning virus. G317A mutants do occur rarely, but itis predicted that A317 will stack on the stem in the same fashion asG317. Further, there are very few non-conservative variations in the NCdomain. Thus, targeting the NC-SL3 interaction for drug interdictionholds special promise in the inability of the virus to escape anti-NCdrugs by mutation.

EXAMPLES Example 1 Affinities of RNA Loops for NCp7

In spite of progress in defining the packaging signal, we havecharacterized the stoichiometry and affinity of NC proteins for the RNAstem-loops only recently (Shubsda, M. F. et al. (2002) Biochemistry41:5276-82). Part of the problem was that most early studies neglectedthe salt dependence of the interaction between the highly chargedcomponents (NCp7 has a charge of +9 at neutral pH, and RNA has onenegative charge per phosphate). Using an ionic strength of 0.2 M reducednon-specific binding and led to full quenching of Trp-37 fluorescence, a1:1 stoichiometry for each of the component stem-loops in the majorpackaging domain (Shubsda, M. F. et al. (2002) Biochemistry 41:5276-82;FIG. 3), and gave results consistent with NMR-based structures of SL3and SL2 complexes (DeGuzman, R. et al. (1998) Science 279:384-388;Amarasinghe, G. K. et al. (2000) J. Mol. Biol. 299:145-156; Amarasinghe,G. K. et al. (2000) J. Mol. Biol. 301:491-511). We found thatnon-specific interactions contributed heavily to the binding at lowionic strength where most previous studies had been done (0.2 M NaCl isnear physiological conditions; in blood the ionic strength is ˜0.18 Mignoring contributions of charged macromolecules; Kratz, A. &Lewandrowski, K. (1998) New Eng. J. Med. 339:1063-1072).

We found that the G₂-loci noted in FIG. 3 are indeed sites forinteraction with NCp7. They form complexes that have dissociationconstants, K_(d)=20-300 nM at 0.2 M NaCl (Shubsda, M. F. et al. (2002)Biochemistry 41:5276-82). Variations in affinity occur among the loopsequences, with SL3 and SL2 binding most tightly (See Table 1 forselected K_(d) values and FIG. 3 for the loop definitions). The bindingconstants are derived from a tryptophan fluorescence assay illustratedin FIG. 5 (hereinafter, “Trp assay”, “Trp-binding assay”, or similarvariations).

TABLE 1 Dissociation constant and relative affinity for RNA-NCp7complexes. RNA K_(d) (nM) RA^(a) SL1a 100 ± 10 28% SL1i 140 ± 20 20% SL223 ± 2 120% SL3 28 ± 3 100% SL4 320 ± 30 9% SL3-UUCG ~7,500 0.4%SL3-GAUA ~16,000 0.2% SL3-GGUG 10 ± 2 280% SL3-all-DNA 230 12% SL3-(DNAstem)-(RNA loop) 30 100% ^(a)Affinity for NCp7 relative to SL3.

The assay is based on quenching of the fluorescence of tryptophan-37 inthe protein by residues in the single-stranded RNA loops. The G318/W37stacking in the SL3-NCp7 structure is illustrated in FIG. 4. Tightlybound RNA molecules quenched nearly all the fluorescence of NCp7 in 0.2M NaCl. The tightest binding variant we have examined (Paoletti, A. C.;Shubsda, M. F.; Hudson, B. S.; Borer, P. N. (2002) Biochemistry 41,15423-15428; “Affinities of the HIV-1 Nucleocapsid Protein for Variantsof SL3 RNA.”; incorporated in its entirety by reference thereto),SL3-GGUG, has a limiting fluorescence that plateaus at the background ofthe buffer (A319 is changed to U in this variant, see FIG. 3). Itsbinding profile approached that of a 1:1 complex with an infinitebinding constant (1/K_(d); similar to the short-dashed line in FIG. 5).In contrast, when the GGAG-tetraloop of tight-binding SL3 is replacedwith UUCG or GAUA, quenching was almost nil indicating very lowaffinity. This is consistent with the primary event in W37 quenchingbeing the close stacking of G318. In addition, there are primarilyelectrostatic interactions between the N-terminal 3-10 helix and the RNAstem (see FIG. 4). The binding site covers a substantial part of thissmall protein's surface.

A 154mer construct (the entire sequence in FIG. 3 that includes all fourstem-loops; SEQ ID NO: 9) bound tightly to NCp7. The assay indicatedthat the equivalent of three NCp7 molecules were bound with highaffinity per RNA (see FIG. 5, where the binding isotherm intersects theaxis at R_(t)/L_(t)≅0.33; it is possible that two strong sites andseveral weaker ones combine to give the appearance of three strongsites). This is the first evidence that multiple NC-interactions arelikely to occur with the 5′-leader.

All but one of the earlier studies of stoichiometry and affinity wereperformed at ionic strengths below 0.2 M (Damgaard, C. K. et al. (1998)Nucleic Acids Res 26:3667-76; Shubsda, M. et al. (1999) Biophys Chem76:95-115; Shubsda M. F. et al. (1999) Biochemistry 38:10147-57;Shubsda, M. F. et al. (2000) Biophys. Chem. 87:149-65; McPike, M. P. etal. (2001) Biochemistry; Amarasinghe, G. K. et al. (2001) J Mol Biol314:961-970; Berglund, J. A. et al. (1997) Nucleic Acids Res 25:1042-9).However, we found unusual properties in the system at low ionicstrength. This included irreproducibility of the binding isotherms,decreases in the initial fluorescence, increases in the residualfluorescence, and binding curves that can only be described by at leasttwo binding constants. The low-salt regime appears to be dominated bynonspecific interactions between these highly charged molecules; thefree protein may also be less structured at low salt. By contrast, SL3titrations at 0.2 M NaCl (FIG. 5) were highly reproducible. The K_(d)for SL3 in Table 1 is the average of 11 determinations with sixdifferent protein preparations (standard deviation <10%).

We found a linear salt dependence at 0.2-0.8 M NaCl (Shubsda, M. F. etal. (2002) Biochemistry 41:5276-82), and estimated that there are fiveto six ion pairs in the SL3-NCp7 complex. In agreement, the NMRstructure predicts six salt-bridge interactions between basicside-chains of the protein and the RNA phosphates (DeGuzman, R. et al.(1998) Science 279:384-388).

SL3 variants and modified RNAs We have studied the sequence dependenceof binding in about 50 variants of SL3 (Paoletti, A. C.; Shubsda, M. F.;Hudson, B. S.; Borer, P. N. (2002) Biochemistry 41, 15423-15428). Thereare 64 possible variants of the loop positions GXYZ using A, C, G, U(the first G-residue was held constant as it is not involved in theloop-NC interaction). Strong binding occurred when XYZ=GNG, but theother loop sequences were considerably weaker. Interestingly, thepreferred sequence for greater affinity was that Z=G; this correspondsto G320, and is not the base that stacks on W37 (FIG. 4). A DNA 20merversion of SL3 had about 15% of the affinity of SL3 RNA.

Several changes are tolerated in the sequence that are useful indesigning the luminescence-quenching pair RNA/DNA chimeric switches ofthe present invention, or in designing test competitors with reducedcomplementarity to the switches (Paoletti, A. C.; Shubsda, M. F.;Hudson, B. S.; Borer, P. N. (2002) Biochemistry 41, 15423-15428). Forinstance, a 16mer RNA construct was found to bind NCp7 with the sameaffinity as the 20mer used in earlier studies (Shubsda, M. F. et al.(2002) Biochemistry 41:5276-82), and only the loop needs to be RNA forefficient binding (see last entry in Table 1). There was virtually nochange in K_(d) upon substituting base pairs near the loop,C316-G321→G316-C321, G315-C322→(C315-G321 or A315-U322). Also,G320→dG320 was well tolerated, and only slight reduction in affinity wasengendered by substituting inosine at loop sites 317, 318, or 320.

We also studied several nucleotide DNA oligomers containing the G-X-Gsequence. These have been reported (Vuilleumier, C. et al. (1999)Biochemistry 38:16816-25; Fisher, R. J. et al. (1998) J Virol 72:1902-9)to possess relatively high-affinity for NCp7. However, none of thesemolecules had even micromolar affinity for NCp7 at 0.2 M NaCl (Paoletti,A. C.; Shubsda, M. F.; Hudson, B. S.; Borer, P. N. (2002) Biochemistry41, 15423-15428).

We mapped the affinities of the wild-type interaction sites for NCp7 inthe major packaging domain of the 5′-leader RNA (Shubsda, M. F. et al.(2002) Biochemistry 41:5276-82), and explored the diversity ofinteractions using variants of the SL3 loop. In order to design andevaluate anti-NC drugs it is useful to know the affinities the proteinhas for its natural substrates under physiological conditions, and toprobe the nature of binding loci by systematic variation of thesequence. The results add to our understanding of RNA-proteininteractions, highlighting problems that may occur when these studiesare conducted at low ionic strength. We also demonstrated that multipleNCp7 proteins interacted with the major packaging domain, and that alinear G-X-G loop sequence in the RNA was not required for highaffinity. A close correlation was found to exist between structuralfeatures and our rapid technique to evaluate the diversity of RNA-NCinteractions.

The Trp-binding assay provided a reliable method to establishstructure/free energy relationships. The simple expedient of comparingaffinities at 0.2 M NaCl is sufficient to distinguish trends that arehelpful in designing anti-NC agents. However, significant obstaclesprevent use of the Trp-assay as a high throughput screen. (1) It is notsensitive enough to accurately measure the affinities of tight-bindingcomplexes, where low concentrations are required for appreciabledissociation of the complex. (2) The Trp assay requires fluorescenceexcitation in the UV., which will restrict its application inhigh-throughput screening of anti-NC drug candidates. (3) A Trp-basedassay is inherently less sensitive than one using dyes for labelingproteins and nucleic acids with lumiphores that absorb and emit in thevisible region of the spectrum.

High-Resolution Structures

The structure of a relevant complex is an extremely valuable guide inundertaking drug-design. The structure for the NCp7-SL3 complex ispresented in FIG. 4. G318 interacts in a hydrophobic cleft of F2, theupper zinc finger shown in the figure and a very similar interaction ismade between G320 and F1. These two residues comprise the G₂-locus forthe SL3 loop, and each G-base makes identical H-bonds with backboneamides and carbonyls in the fingers. Electrostatic interactions alsoplay a role because of the high formal charges involved, +9 for Zn₂.NCp7and −19 for the SL3 20mer. The electrostatic surface of the protein (notshown) has the RNA in a deep electropositive pocket on thenucleotide-binding surface of the protein. We also used NMR to determinethe structure of the unbound RNA, which alters considerably upon bindingthe protein (Pappalardo, L. et al. (1998) J. Mol. Biol. 282:801-818).The structures of the finger domains are largely determined bycoordination to the zinc, and do not change upon binding RNA or DNA.However, the linker and the termini are flexible in the absence ofnucleic acid at low ionic strength (Lee, B. M. et al. (1998) J. Mol.Biol. 279:633-49). The N-terminal residues form a 3-10 helix in thecomplexes with SL3 and SL2; this helix interacts mainly by salt-bridgeinteractions with the RNA stem.

High-Throughput Assays

Our work on the RNA-NC complex is relevant to the design ofhigh-throughput drug discovery. We have established reliable assayconditions, and describe a multiplex assay to examine many thousands ofpotential inhibitors. We also have completed a survey of the affinitiesof the most important wild-type RNA substrates for NC binding, and haveexplored the diversity of interactions (Table 1). We havehigh-resolution structures to guide our search for anti-NC agents.

The design of high-sensitivity and high-throughput assays take advantageof our earlier work on the SL3-NCp7 system. The detection scheme useshighly efficient fluorophore labels similar to known molecular beacons(Tyagi, S. & Kramer, F. R. (1996) Nat Biotechnol 14:303-8; Fang, X. etal. (2000) Anal Chem 72:747A-753A). The beacons utilize the processcalled Fluorescence Resonance Energy Transfer (hereinafter “FRET”).

Example 2 Unimolecular Biostable Nucleic Acid Switch

The principles of the unimolecular bistable OrthoSwitches areillustrated in FIG. 6 and SEQ ID NO: 7. The DNA part of the sequence(shown in red) was designed to make the “H” state be more stable thanthe two-headed “O” state. The high affinity NC-binding sequence, GUG, islocated in an RNA tetraloop in the O-form. The DNA sequence wasengineered to set the equilibrium constant, K1, between 0.01 to 0.1 and,as shown below, it is 0.08. This simply means that the ratio ofconcentrations, [O]/[H]=0.08. A fluorophore is placed at the 5′-end ofthe chain (*), and a quencher at the location marked Q. The distancebetween fluorophore and quencher is much larger in H than in O, so H hasmuch stronger fluorescence. Such “molecular beacons” operate on theprinciple of FRET. The efficiency of transfer increases as the inversesixth quencher. Thus a small change in distance between the dyes canlead to a large change in fluorescence.

The scheme in FIG. 6 a is now easily understood. The K1 equilibriumfavors the “bright” H-form in the absence of NC protein, P. However,increasing [P] will cause it to bind GUG, consuming O. As more P isadded, H is consumed to produce more of the “dark” P.O complex. However,a drug candidate, C, that binds strongly to P will displace O, whichreverts to the bright H-form. A multiwell format for high throughputscreening would be to put identical amounts of switch and protein ineach well, then to add a different candidate to each. The wells that arebrightly fluorescent contain promising drug leads with substantialaffinity for the protein target.

Tests show the system works as planned. The fluorescence decreases as NCis added, and a minimum fluorescence for the O-form can be establishedby adding ribonuclease. These features are clearly seen in FIG. 7. Theheavy blue curve shows the fluorescence emission spectrum of a 10 nMsolution of C3 (SEQ ID NO: 7). Increasing amounts of NC were added,resulting in a rapid decrease in fluorescence as the H-form is consumedto make the P.O complex. This titration with more data points is shownin FIG. 8.

Example 3 Screening for NC Inhibitors

The NC/switch complex is capable of identifying a high affinityinhibitor of NC by generating an optical signal. This requires theprotein bound “dark” switch to dissociate in the presence of a highaffinity NC inhibitor, producing an unbound “bright” switch.

In this experiment, SL2 from the HIV-1 packaging domain was used. It hasa high affinity for NC (K_(d)=25 nM) and is not predicted to bind to anypart of the switch at the concentrations used. SL2 also has GUG in theloop, similar to the O-form of the NC-Switch, but has a very differentstem sequence.

FIG. 9 illustrates the results. The first bar shows the fluorescenceintensity of the labeled C3 switch. The second bar shows the intensityafter adding enough NC to switch a considerable fraction to the darkO-form. Addition of an amount of the SL2 inhibitor equal to the NC addedproduced a nearly instant response from C3 that is released from the P.Ocomplex. The intensity increases again in the expected fashion. Thedegree of the increase is consistent with the equilibrium constants.

Another experiment is illustrated in FIG. 10. Here, the competitor andNC-switch are premixed, and NC is added. SL2 and C3 were added to acuvette and the C3 fluorescence was monitored as a function of [NCp7].SL2, which has a higher affinity for NC, out-competes C3 for NC binding.This competition appears as an initial plateau in quenching (FIG. 10).This experiment also demonstrates that a high affinity NC competitor caninterrupt formation of the switch-NC complex. The intensity decrease isagain consistent with the equilibrium constants.

We also showed that the switch does not respond to the addition ofbovine serum albumin at twice the concentration of NC used in theseexperiments. Neither does the switch respond to the addition of tapwater, nor to water that contains human saliva (not shown).

Example 4 Luminescent Nucleic Acid Switches

FIG. 11 shows the equilibria for the SL3-NCp7 assay using a tetheredluminescent switch in accordance with the first embodiment of thepresent invention. The outline of a luminescence assay used to detectcompetitors of SL3-NCp7 complex formation is shown in the inset (topright). The sequences denote a tethered switch with two stable states:one where the binding site (GGUG sequence) is hidden, H, at left, andone where the binding site is open, O, at right. RNA segments aredenoted in dark italics, DNA in lighter font. Tethering is accomplishedby the fifteen base-pair stem that does not vary between the two statesof the switch. This “fastener” stem is fixed, i.e., its sequence doesnot vary in optimizing the performance of the switch. The probe segment,P, is embodied in the RNA sequence, G18-C33, in the chimeric strand. Thecover segment, C, is embodied in the sequence, G1-C16, of the all-DNAstrand. L=Ligand, C=competitor, *=lumiphore, Q=quencher, D2=locationfor * if it is desired to make the signal of O-form highest and theH-form lowest.

The FRET system outlined in FIG. 11 can be configured to detect thepresence or absence of NC-RNA complexes, and can be adapted to nearlyany other protein-RNA or protein-DNA complex. For the SL3-NCp7equilibrium, the SL3 RNA hairpin is available at the right of theRNA/DNA chimeric strand in the O-state. This switch has fluorescentlabels, * (e.g., FAM, 6-carboxy-fluorescein), at position 17 of theall-DNA chain and Q (a quencher, e.g., dabcyl) at the 5′-end of the DNAsegment (light font). Any luminescent label and quencher now known inthe art may be used in the switches of the present invention. In the Ospecies, * and Q are within the Förster distance for efficientquenching. However, they are far apart in the H-form, and the switchemits strongly.

By altering the DNA sequence, K₁ is adjusted between 0.1-0.01(K₁=[O]/[H]). This gives 90-99% of the maximal luminescence signal inthe absence of the other components. Upon addition of NCp7 (L), the K₁equilibrium shifts to the right, and the luminescence signal decreases.The switch is set to a minimal signal in the presence of a slight excessof L, and is triggered to emit when a competitor, X, sequesters theprotein in the L.X complex. The luminescent nucleic acid switches of thepresent invention share some properties with the “scorpion” probes usedin real-time PCR applications (Solinas, A. et al. (2001) Nucleic AcidsRes 29, E96; Thelwell, N. et al. (2000) Nucleic Acids Res 28:3752-61).Knowledge of K₁, K₂, and the input concentrations, O_(t), L_(t), andX_(t) will allow estimation of K₃.

The bistable nature of the free switch and switch-protein complex isrelated to thermodynamic properties. The populations of the species willreach an equilibrium state that can be predicted with confidence fromthermodynamic databases (Sugimoto, N. et al. (1995) Biochemistry34:11211-6; SantaLucia, J. (1998) Proc Natl Acad Sci USA, 95, 1460-1465;Mathews, D. H.; Sabina, J.; Zuker, M.; Turner, D. H. (1999) J Mol Biol288, 911-940; Zuker, M. (see web site: bioinfo.math.rpi. edu/˜zukerm).The procedure is analogous to using two entries in the free energytables in a physical chemistry text to predict the equilibrium constantfor a third reaction. While such tables in chemistry texts are oftenaccurate to 0.01 kcal/mol, the DNA/RNA databases have uncertainties onthe order of 1 kcal/mol. That is enough to change the populations of thefree and protein-bound switches by nearly an order of magnitude.However, as shown in Table 2, a few changes to the sequence can changeK1 by twelve orders of magnitude. Therefore, one of skill in the artcould readily fine-tune the equilibrium constant K1 by changing thesequence, and then monitoring the populations of the two species bymeasuring luminescence in accordance with the present teachings.

A multiwell, array or microarray format may be applied in accordancewith one preferred embodiment of the present invention to screensmall-molecule inhibitors for their potential to bind NCp7 orNC-containing precursors. It can be determined whether a compoundpermanently inactivates L or O, or releases the lumiphore by hydrolysisof the nucleotide chain. Titrations or bracketing tests can be used toclassify “hits” in these screening assays. Hits will also be subjectsfor a “minus L” control; interference from competitor-nucleic acidinteractions might be apparent from adding competitor to a luminescentswitch with K₁=10-100. Changing the location of the quencher to the3′-end of the RNA segment can also be used to make confirmatory tests.In that case, the O form is highly luminescent, and the assay will showa null for effective competitors.

Embodiments of the invention are directed to bistable A1/A2-constructscan be designed to detect competitors for other A2-L or A1-L complexesother than the HIV-1 nucleocapsid protein. Applications includeRNA-protein interactions where the RNA binding site can be designed intothe O-form. These include the RRE-rev and TAR-tat RNA-protein complexesin HIV-1. Other competitors of naturally occurring DNA-protein orRNA-protein complexes can be designed where the favored binding site forL occupies an analog of the O- or H-form. This design feature of thepresent disclosure distinguishes it from approaches based oncombinatorially-derived sequences. However, the use of empiricallychosen, rather than engineered, ligand binding domains is not precluded.

RNA or DNA molecules referred to as “aptamers” can be selected to bindnearly any protein or other molecular target (Jayasena, S. D. (1999)Clin. Chem. 45:1628-1650). An aptamer or other combinatorially-derivedsequence binding site can be included in the A1- or A2-form, as well.The use of combinatorial technology to develop high affinity DNA- andRNA-protein binding sites represents an alternative to naturallyoccurring DNA- and RNA-protein binding sites described above. It iscontemplated that high-throughput screens based on our invention can bedeveloped for a wide array of therapeutic targets, remediation ofbioterror agents, and other applications. It is also contemplated thatsensitive and specific ligand-detection assays based on our inventioncould be developed for a wide array of proteins, nucleic acids,saccharides, toxins, and other molecules of diagnostic importance inhumans, animals, plants, and other organisms, as well as for nearlyinstantaneous and specific assays for bioterror agents, and otherapplications.

Simple modifications to the scheme just described are required for thediagnostic applications. A competitor, X, does not need to be presentfor most diagnostic applications, in which case the K3 equilibrium inFIG. 11 is removed from consideration. The ligand-detection assay shouldgive a minimal signal in the absence of L, and a strong signal whenthere is a substantial amount of the A2-L bound complex. One suitablealteration moves the luminescence donor, *, to the right-hand end of thechain shown in FIG. 11 (position D2). Then the H-form has a minimalsignal because a lumiphore is near in space to a quencher. By contrast,the O-form is highly luminescent because the quencher is situated beyondthe distance for efficient suppression of the signal. Other arrangementsof lumiphore and quencher can be contemplated that would be useful todetect the state change.

Design Parameters for Luminescent Nucleic Acid Switches

The NC binding site. A basic design feature illustrated in FIG. 11 is toprovide the highest affinity G₂-locus in the RNA loop with a stemidentical to SL3; this feature is present at the right side of O in thefigure. At the same time, the DNA and RNA hairpin loops arecomplementary to each other; GGUG will be double-stranded in H, andtherefore unavailable to bind NC. The single-stranded DNA loop in O willnot compete for NC, as we have shown that C-rich loops, and DNA loops ingeneral, have much lower affinity (˜10,000 times less for these d(CACC)loops). Note that we have chosen the GGUG loop sequence, which bindsNCp7 more tightly than the wild-type GGAG (see Table 1; K₂ and K₃ aredissociation constants for the relevant complexes in this text).

Tuning the K₁ equilibrium. The switch design will preferably aim for0.01<K₁<0.1 in the luminescence assays. A small K₁ value favors the“bright” H conformation, and will give a near maximal signal-to-noiseratio (S/N) when the system switches from the “dark” LO form. However,K₁ should not be too small or an extremely high affinity K₂ will benecessary to switch to LO. That is because the second equilibrium isconcentration-dependent, and O_(t) and L_(t) will be ˜0.1-10 μM.Preferred S/N will result if [H]/([O]+[LO])>3 at reasonable L_(t) valuesin the presence of an interesting competitor. Thus, when K₂ is ˜10-20nM, K₁ is preferably set to trip the switch from off to on over a smallincrease in X_(t).

The K₁ equilibrium is adjusted by changing the sequence of the all-DNAstrand, particularly near positions 4, 8, or 13 (see FIG. 11). Whenthese residues are all complementary to their RNA counterparts, H hasfour base pairs more than are present in the DNA and RNA stems of O.Then H will dominate the K₁ equilibrium. The stabilities, ΔG° (H) andΔG° (O), can be estimated from thermodynamic databases (Sugimoto, N. etal. (1995) Biochemistry 34:11211-6) for forming a folded structure ofDNA, RNA, or a DNA/RNA hybrid from the unstructured coils. Thedifference in free energy for the two forms is ΔG₁°=ΔG° (O)−ΔG° (H)since their unstructured reference states are identical. We haveestimated the free energy for 22 sequences and find that by forcingmismatches in H that are compensated in O, K₁ can be varied over 12orders of magnitude. Several of these put K₁ in the preferred range forthe applications disclosed herein. It is be within the ability of aperson skilled in the biophysical chemistry of nucleic acids toconstruct an O

H system with the desired properties starting from the disclosedpredictions.

Table 2 provides guidance to the skilled practitioner in calibrating theK, equilibrium. The bright state, H, has been characterized as the fullypaired 40mer, M1, with * and Q as in FIG. 11 b, and the properties ofthe dark state were demonstrated by the M2 molecule, which favors 0 by afactor of ˜4000 over H. The latter has K₂ similar to that of SL3 RNA;this was tested using the Trp-assay. The luminescent switches, M2-M5,should be bright in the absence of NCp7 and dark in its presence (thishas been verified for M2). Each of the molecules disclosed herein hasbeen tested with MFOLD (see web site: bioinfo.math.rpi.edu/˜zukerm) toensure that no alternative secondary structure will be present. Notethat the lengths of the base-paired stems, exact positions of thelumiphore and quencher, and detailed sequence can vary from thatpresented for M1 through M5, and still fall within the bistable H/Oclassification encompassed within this disclosure.

TABLE 2 Predicted K1 and mismatch sites for four molecules compared tothe fully paired H chimera, M1* POSITION M1 M2 M3 M4 M5 3 T A 4 A T T 5G T 8 A T 9 C AAA 12 C A 13 T A A 14 A T K₁ 6 × 10⁻⁹ 4 × 10³ 0.08 0.0030.002 *Blank entries signify that the site has the same base as in M1;the full M1 sequence is given in FIG. 11.

The full sequences for the all-DNA chains in the M1-M5 tethered duplexes(SEQ ID Nos. 1-5, respectively) are:

(SEQ ID NO: 1) M1_DNA = dabcyl-d(GCTAGCCACCGCTAGC(T-FAM)-CACAGCACGACTCAGATGG); (SEQ ID NO: 2) M2_DNA= dabcyl-d(GCATGCCTCCGCATGC(T-FAM)- CACAGCACGACTCAGATGG); (SEQ ID NO: 3)M3_DNA = dabcyl-d(GCTATCCACCGATAGC(T-FAM)- CACAGCACGACTCAGATGG); (SEQ IDNO: 4) M4_DNA = dabcyl-d(GCTTGCCACCGCAAGC(T-FAM)- CACAGCACGACTCAGATGG);(SEQ ID NO: 5) M5_DNA = dabcyl-d(GCTAGCCAAAACGCTAGC(T-FAM)-CACAGCACGACTCAGATGG);

The DNA/RNA chimeric chain is constant in each of the tethered duplexes,M1-M5, having the full sequence;

(SEQ ID NO: 6) d(CCATCTGAGTCGTGCACGC)-UAGCGGUGGCUAGC;

The DNA residues are denoted by “d(XXX)” and RNA residues are notenclosed by parentheses; sequence alterations from M1 (also shown inFIG. 11) are underlined. The fluorophore(FAM=6-carboxymethylfluorescein) and universal quencher (dabcyl, methylred) are well-known to practitioners skilled in FRET technology. Thedabcyl label can be attached via a 5′-phosphate at the 5′-end of the DNAchain, T-FAM derives from the replacement of the 5-methyl of T by FAM;both dyes can be incorporated via ordinary solid-phase coupling ormodification after solid-phase synthesis using standard methods.

In some embodiments the databases can predict only the order ofmagnitude of K₁. Therefore, experiments with two to four mismatchedvariants may be required to tune the assay for optimal switchingperformance. It is likely that the order of free energies predicted fromthe databases is correct, even if the actual values are not. Forinstance, if K₁ is found to be too small for best performance, anall-DNA strand predicted to favor O more strongly can be substituted.

Annealing strands tethered by duplexes. The strands of a switch that aretethered by a fastener duplex may be annealed to each other prior toconducting experiments related to the detection of ligand or acompetitor binding. This can be conducted by standard methods familiarto one skilled in biophysical chemistry. A suggested process is to mixequimolar concentrations (near 1 micromolar) of strands at an ionicstrength between 0.1-0.5 M, pH between 6-8. The strands can then beannealed by slowly reducing the temperature from 80° C. to roomtemperature over a period of 10-20 min.

Equimolar mixing of strands will be accompanied by maximum luminescenceif the signaling arrangement described in FIG. 11 is used. A 5% molarexcess of the DNA/RNA chimeric strand over the all-DNA strand in FIG. 11will not affect most molecular switching applications, as ligandcomplexes with this chimeric strand will produce no signal and competeto an extent <5% with the tethered switches. A 5% molar excess of theall-DNA strand over the chimeric strand may result in a backgroundluminescence signal that is higher than for a 1.00:1.00 mixture ofstrands. However, unannealed all-DNA strands will not form ahigh-affinity complex with the ligand, so these isolated strands willnot compete effectively with the tethered switches.

Some applications may benefit from a tethering duplex, F, longer orshorter than the fifteen base pairs depicted in FIG. 11. In addition toestimating the free energy of duplex formation (ΔG°_(f)) using thestandard thermodynamic databases, it is prudent to measure ΔG°_(f) usingthe DNA strands that compose F. In the example posed by FIG. 11, thestrands, d(AGCACGACTCAGATGG) and d(CCATCTGAGTCGTGCA) encompass F and“dangling” single-stranded A residues that contribute to stabilizing theduplex against strand dissociation. ΔG°_(f) for F can be measured underthe ionic strength and pH conditions contemplated for use of the switch.The equilibrium constant for duplex formation can then be calculated atthe concentration of switch contemplated for use of the switch.Absorbance vs. temperature profiles, calorimetry, or other experimentaltechniques can be used to measure ΔG°_(f). This process is familiar toscientists skilled in the biophysical chemistry of nucleic acids.

One could have a concern that the hairpin-forming elements of theswitches might dimerize. However, when the dimerization equilibriumconstant is estimated from the thermodynamic databases, and theconcentrations for the luminescence applications are used, the amount ofsuch dimers is vanishingly small.

Simulating the binding equilibria. Optimizing performance of the assaysand analyzing the results may be greatly assisted by simulations withinput values of K₁, K₂, K₃, O_(t), L_(t), and X_(t). The coupledequilibria describing all of the species result in a cubic equation forthe luminescence competitor assay. This requires special treatment tosolve for roots that are physically reasonable and that allow continuousvariation of X_(t) or other species in simulating titrations (Press, W.H. et al. Numerical Recipes, Cambridge U. Press, New York, 1986).

FIG. 12 illustrates the performance characteristics of a high throughputscreening assay. At the dashed vertical line, X_(t)=10 μM, which isoften used for testing libraries of chemical compounds. The simulationsare helpful in adjusting the assay conditions for lower orhigher-affinity competitors, other concentrations of competitors, etc.

Dynamic range is a function of the input variables K₁, K₂, O_(t), L_(t),and X_(t). Under the conditions at the dashed line in FIG. 12, a dynamicrange of 100 in K₃ can be distinguished in one well of a microtiterplate. Other wells can have other values for the input parameters. Adynamic range of 10,000 is reasonable for two or more wells per sample.

For low-affinity inhibitors, high concentrations are required andsensitivity is not an issue. Sensitivity is important for luminescenceassays of the present invention only when the inhibitor affinity is sohigh that very low protein and inhibitor concentrations are needed toforce appreciable dissociation of the complex. At that point, it is notnecessary to measure an exact K_(d)-instead, one may need ADME/Toxassays to determine whether this is a bona fide drug lead.

Synthetic considerations. The luminescent chimeric switches tethered byfastener duplexes are made in two pieces. The “left-oligo” (left side ofthe H and O forms in FIG. 11) is composed exclusively of DNA andcontains both the * and Q labels. The right-oligo contains both DNA andRNA and the D2 luminescence donor only for switches that are designed togive maximal luminescence upon binding the ligand. We have found thatonly the SL3 loop needs to be RNA for efficient NCp7 binding (last entryin Table 1). Increasing the number of DNA bases, especially at the endsof the chains (away from the ligand binding site), increases thestability of switches against contamination by ribonucleases. Currentsolid-phase synthesis technology can produce 30-50mer DNA, RNA, andchimeric strands in high yield. Signaling entities, such as FAM, dabcyl,etc., can either be added during solid-phase synthesis by incorporatingthe appropriately protected phosphoramidite, or by common proceduresthat add the labels after solid-phase synthesis is complete.

It is routine to purify 20mer-40mer length molecules by anion-exchangeHPLC. Reversed-phase HPLC is also useful for purifying luminescentlytagged oligomers from unlabeled versions. In some cases, especially withmolecules that are >30mers, it is desirable to purify molecules bypolyacrylamide gel electrophoresis (PAGE). Non-denaturing PAGE and Gelfiltration chromatography are useful in purifying tethered duplexes fromisolated single strands.

The extra flexibility in assembling the duplexes will prove valuable.The RNA side has only one sequence. It will be useful to have manysequences available for the all-DNA side to facilitate calibrating theK₁ switch at different levels. This is also useful in rankingcompetitors in several categories that differ in K₃.

Based on fluorescence quantum yields, the signal from the fluoresceinanalog, FAM, in the switches of the present invention are 20-50 timeslarger per photon absorbed than for our standard Trp-assay. A visiblewavelength laser can be used to excite FAM, whereas a lower intensity UVlamp must be used for Trp. Detection also has a reduced background forvisible compared to UV emission. Therefore, the signal/noise ratios forunquenched FAM will be 100-1000 times larger than for unquenched Trp.This sensitivity is comparable to ³²P-labeling, without radiationconcerns or the approximations that are required to interpretfilter-binding assays.

It is appropriate to examine the two extreme states in FIG. 11(sequences M1 and M2 in Table 2). This allows evaluation of the positionof the K₁ equilibrium shift upon changing the sequence. Simply put, theluminescence of pure H and O are known, so their populations in a mixedsystem are just linear functions of the measured luminescence.

Improvements may be effected by using a construct that brings the * andQ labels closer together in the O-state, or introduces afluorophore-quencher pair that more efficiently suppresses fluorescenceat short distances (Integrated DNA Technologies, (www.idtdna.com); Coty,C. (2002) Drug. Discovery & Development 5: 44-51; (www.nanoprobes.com).Even at a 2-fold difference in fluorescence between H and O, thesensitivity of the experiment is far higher than for the Trp-quenchingassay. Another possible mode of the present invention is to use thedifference in fluorescence lifetimes of the free and protein-boundswitch. One expects the fluorophore to have a longer fluorescencelifetime when bound to NC than when it is free in solution. With phasemodulation methods (modulated light intensity and a lock-in amplifier)one can get a steady state signal that attenuates the contribution fromthe short time component. There are commercial applications of phasemodulation technology in high-throughput screening (Kashem, M. A. (2001)5^(th) Intl. Drug Disc. Prod. Users Mtg; (www.zymark.com);www.moleculardevices.com). The use of phase modulation in preferredembodiments of the present invention are expected to yield an increasein on/off discrimination of about 100-fold.

Other fluorophore quencher pairs which may be used in accordance withpreferred embodiments of the present invention include those listed inthe Integrated DNA Technologies catalog(www.idtdna.com/program/catalog/DNA_Probes_main.asp) and the MolecularProbes catalog (www.probes.com/servlets/masterlist). Of particularutility in construction of the switches disclosed herein are thefollowing quenchers: dabcyl, BHQ-1, BHQ-2, Iowa Black, and Nanogold, andthe fluorophores, 6-FAM, TET, HEX, Cy3, Cy5, eosin, coumarin, OregonGreen, Rhodamine Green, Rhodamine Red, Texas Red, TAMRA, ROX, JOE,Bodipy dyes. Of course any other quenchers known in the art are alsoconsidered applicable to construction of the fluorophore quencher pairsdisclosed herein.

Others have proposed bimolecular RNA-RNA or DNA-DNA probes that couldproduce similar results to the switches disclosed herein forhigh-throughput screens (Jayasena, S. D. (1999) Clin. Chem.45:1628-1650). However, these applications have been designed such thatthe screen has probes switch between single-stranded and duplexedstates. This brings the unavoidable fact that the kinetics of formingthe complex are second-order, and thus concentration dependent. Forexample, we experimented with a bimolecular complexation system (i.e.,removing the fastener duplex tether in FIG. 11). At the lowconcentrations typically used in fluorescence assays, the time fornearly complete equilibration was ˜10 hr at room temperature. While ahigh-throughput assay for competitors is still possible, it wouldrequire either a long equilibration time, or annealing by heating. Itwould be preferable to avoid heating in the presence of a ligand orcompetitor that might denature. Using untethered bimolecular probes itmay be awkward to perform titrations for determining accurate K₃ values.Simulations may also be more difficult, involving equations that arefifth-order in some of the variables. This may make designing the assaysmore difficult, as well as complicating interpretation of the results.

There is another aspect of kinetics that may be significant in regard tocertain aspects of the present invention. For example, with respect tothe bistable tethered switches of the present invention, the system mustgo through a partially paired intermediate to convert from one state tothe other. There will be an associated activation barrier that slows theconversion between the two forms. The barrier probably depends on thelength of the stems in the hairpins segments. Therefore, we prepared a16mer SL3 construct, which has two base pairs removed from the 20mer SL3stem used previously. The Trp-assay showed that both have virtuallyidentical K_(d) values. The 16mer has been incorporated in a preferredembodiment of the luminescent switch design (FIG. 11), and we have shownthat equilibration with NCp7 has been shown to occur within a minute ata 10 nM concentration of the M3 switch. Another aspect favoring rapidequilibration across K₁ is the chaperoning aspect of NCp7, which allowseven lambda DNA to quickly adjust to its lowest free energy form underforce-induced stretching.

Competitor Binding

The experiments outlined below start with applications of knowncompetitors of the NC-RNA complex then move to those that have thepotential to become useful drugs directed against new anti-HIV targets.

Ligands with known affinity. The Trp-assay cannot determine theaffinities very well for tight-binding ligands of NCp7. Reviewing FIG. 5reminds us that titrations for such ligands will deviate only slightlyfrom the dashed 1:1 line for K_(d)=0. The deviation is largest when thecomplex approaches saturation, so only a few data points control thevalue determined for K_(d). Since the deviation and the S/N are smallfor these points, the effect of experimental errors is large.

The luminescence-quenching assays using the switches of the presentinvention offer a method to determine K_(d)=K₃ by competition, such aswith SL3-GGUG. As shown in FIG. 12, the assay is capable of determiningK₃ accurately. The assay effectively balances K₃ against K₂ and K₁, so atitration provides many high S/N data points. Switch molecules can betested by determining whether they reproduce known K_(d) values. Forinstance, NC-switches can be tested with the RNA molecules listed inTable 1. Good choices would be SL2, SL1a and SL4, which have noappreciable complementarity to any part of the switch.

Measurements of unknown affinities RNA aptamer constructs selectedagainst NCp7 for which nanomolar affinities have been asserted frommeasurements at low salt concentrations (Lochrie, M. A. et al. (1997)Nucleic Acids Res 25:2902-10; Berglund, J. A. et al. (1997) NucleicAcids Res 25:1042-9; Allen, P. et al. (1996) Virology 225:306-15), havebeen studied with our tryptophan assay. The aptamer construct sequenceshave been published, and appropriate RNA molecules are availablecommercially, which were purified by standard methods. Some of theseaptamer constructs do not have obvious G₂-loci, and could be very usefulin expanding our general understanding of the basis for NC-RNA bindingspecificity. However, we have shown that these published aptamerconstructs bind two or more NCp7 proteins per RNA, rendering themmarginally useful in drug discovery applications. Other RNA constructswe have inferred from aptamer sequences have 1:1 stoichiometry andaffinity for NCp7 that is similar to SL3 and SL2.

The cyclic peptide, c(F-C-dW-R-C-K), has been shown to have effects thatsuggest it competes with NCp7 (Druillennec, S. et al. (1999) Proc NatlAcad Sci USA 96:4886-91). This luminescence-quenching assay would not bedone in the competition mode described above. Instead, the quenchershould be located at the D2 position in FIG. 11, and the dominantH-species will be dark. If the peptide binds to 0, the switch will lightand allow measurement of K_(d)=K₂. If the c(F-C-dW-R-C-K) competition isfavorable, other cyclic peptides may be used in accordance with thisembodiment of the present invention.

The competitor binding tests allow us to measure affinities of compoundlibraries in a high-throughput fashion. They also provide a means todetermine whether designed or combinatorial changes improve the affinityof anti-NC candidates.

Other Target Interactions

In addition to the interactions between the viral RNA and the NC domaindescribed above, any other target interactions with RNA, DNA, proteins,precursors, and saccharides may be exploited in accordance with thepresent disclosure. Some of these targets include, without limitation,the internal ribosome entry site (IRES) of Hepatitis C Virus, IRES sitesin other viruses, as well as agents involved in the etiology of viralinfections related to Congo-Crimean hemorrhagic fever, Ebola hemorrhagicfever, Herpes, human cytomegalovirus, human pappiloma virus, influenza,Marburg, Q fever, Rift valley fever, Smallpox, Venezuelan equineencephalitis, and targets in HIV-1, MMTV, HIV-2, HTLV-1, SNV, BIV, BLV,EIAV, FIV, MMPV, Mo-MLV, Mo-MSV, M-PMV, RSV, SIV, AMV, and other relatedretroviruses, including but not limited to: TAR-tat, RRE-rev, DIS, PBS,RT, PR, IN, SU, TM, vpu, vif, vpr, nef, mos, tax, rex, sag, v-src, v-mycand precursors and protease products of the precursors: gag, gag-pol,env, src, one, as collected in Appendix 2 of Coffin, J. M., Hughes, S.H., Varmus, H. E. (1997) Retroviruses, Cold Spring Harbor Lab Press,Plainview, N.Y.). Other targets in bacteria, fungi, insects, and otherpathogens and pests of humans, animals, and plants may also beapplicable to the present switches and methods, including but notlimited to B. anthracis, (especially the components of the toxin:protective antigen, lethal factor, edema factor, and their precursors),Burkholderia pseudomallei, Botulinum toxins, Brucellosis, Candidaalbicans, Cholera, Clostridium perfringins toxins, Kinetoplasts,Malaria, Mycobacteria, Plague, Pneumocystis, Schistosomal parasites,Cryptosporidium, Giardia, and other environmental contaminants of publicand private water supplies, Ricin, Saxitoxin, Shiga Toxin from certainstrains of E. coli, Staphylococcus (including enterotoxin B),Trichothecene mycotoxins, Tularemia, and agents causing Toxoplasmosis,as well as contaminants of food and beverages that may be deleterious tohuman or animal health. The detection and screening methodologiesafforded by some embodiments of this invention may also be applied tosmall-molecule targets, including but not limited to nerve gas agentsand chemical poisons, as well as contaminants of public and privatewater supplies, of food and beverages, and of indoor air that may bedeleterious to human or animal health.

Example 5 Branched Nucleic Acid Switch

Embodiments of the invention are directed to an RNA/DNA chimeric switchwith three branches which include a “probe” segment, P, a “cover”segment, C, and a “toggle” segment, T. P contains one or more highaffinity sequences for a target molecule, C is completely or nearlycomplementary to P, and T is mostly complementary to P, such that alarge population of the switch molecules include C:P stems and a smallerpopulation includes T:C stems. An appropriate combination of signalingentities is attached to the strand termini to conveniently read out therelative populations of the stable states. It is also contemplated thatmultiple switches embodying different probe strands could havecoordinated activities. Applications of the latter may include sensingcascades that amplify the response of a first switch. This could lead tomulti-unit molecular amplifiers, state-switchable nanostructures, and/ormulti-unit molecular nanomachines.

The three-fold connection in FIG. 8 can be made using a “doubler”phosphoramidite originated for the creation of nucleic acid dendrimers(http://www.glenres.com/ProductFiles/Technical/TB_dC_Brancher.pdf; T.Horn and M. S. Urdea, Nucleic Acids Res, 1989, 17, 6959-67; M. L.Collins, et al., Nucleic Acids Res, 1997, 25, 2979-84; T. Horn, C.A.Chang, and M. S. Urdea, Nucleic Acids Res, 1997, 25, 4842-4849; T. Horn,C. A. Chang, and M. S. Urdea, Nucleic Acids Res, 1997, 25, 4835-4841).The central branch can also mimic the branched intermediate in RNAsplicing, where the 2′, 3′, and 5′-OH groups of an RNA nucleotide allbear phosphodiesters connecting to each of the three chains.

FIG. 13 illustrates the embodiment in which P, C, and T are fastened ata single vertex, thus comprising a unimolecular switch. FIG. 13( a)shows a 3-fold junction connecting the P, C, T segments. The designationT_C_P denotes the unpaired random-coil reference state for free energycomparisons. The bistable molecule can exist in either the T_C:P stateor the T:C_P state (FIG. 13( b)). The ligand binding site in P issequestered in T_C:P and available in T:C_P. The population of T_C:P ishighest in the absence of ligand binding. The distances between thesignaling entities, S, S′, and S″, change when the switch changes state.

A free energy diagram depicts relative and barriers to interconversionof states (FIG. 13( c)). In the absence of binding to the ligand, X, thelow free energy form has P sequestered in the C:P complex. When T:C_P isnot bound to X the switch has a higher free energy. The free energy ofthe T:C_P:X complex decreases as the concentration [α]increases, tippingthe populations in favor of the T:C_P form of the switch.Interconversion between the two forms is slow if the intermediate formis similar to T_C_P, where most or all of the base pairs are broken. Thetwo forms convert rapidly if the intermediate steps occur via branchmigration, where only a few base pairs are broken in a helix defect thatmigrates from one end of the C segment to the other.

The sequences of the three segments can be engineered to create anefficient molecular switch (FIG. 13( d)). A “1” denotes a nucleotidecomplementary to a “2”, and “3” is complementary to “4”. The “combimer”contains the ligand binding site, in P, which is written 5′-3′, left toright. The cover segment, C, written 3′-5′, can be slightly longer thanP, but otherwise is depicted as fully complementary. The toggle segment,T, is depicted as having a mismatch with C in the combimer zone, but isotherwise fully complementary. Several trials can be made to evaluatethe effect of different base pairs and mismatches using thethermodynamic databases. This is necessary to ensure optimal performanceof the switch by setting K1 at around 0.01 to 0.1 as in FIG. 11.

Analogs of Branched Nucleic Acid Switches Using a Tethering Duplex

FIG. 14 illustrates three embodiments which are analogous to FIG. 13 b.As in the embodiment illustrated in FIG. 11, the fastener stem, F, isstable and does not substantially alter during switching events. P, C,and T have the same meaning as in FIG. 13. Locations of signalingmoieties, S, S′, and S″ can be optimized to create the most robustsignal output. FIG. 14 c resembles the secondary structure in a singlechain of covalently attached residues formally known as a “pseudoknot.”

FIG. 15 illustrates an embodiment in which P and C lie on separatestrands, which are held together by stable fastener stems, F1 and F2.The construct in FIG. 11 has a similar appearance, with F2 beingzero-length. The designation C_P denotes the open conformation where theprobe segment is available to bind the ligand. The ligand binding sitein P is sequestered in the C:P state. The folded form at the right isformally known as a cruciform structure. The distal ends of F1 and F2may be joined, in which case P and C reside in a covalently closedcircle that may be supercoiled. The density of supercoils can influencethe C:P⇄C_P equilibrium.

Supercoiling in DNA is well-known (Cantor, C. R. & Schimmel, P. R.(1990) Biophysical Chemistry Part III, 1265-1290). It is easy to controlthe density of superhelical turns using intercalating dyes andtopoisomerase enzymes (Wang, J. C. (1996) Annu. Rev. Biochem. 65:635-92;Wang, J. C. (1984) J. Cell Sci Suppl. 1:21-29). The helical nature ofdouble-stranded DNA engenders a substantial resistance to supercoiling;this resistance can be tapped to switch the state of a localized domainto reduce the superhelical stress. The resistance can be quantified by afree energy, ΔG° (sup), which expresses the ability to cause chemicalchange upon relaxing the superhelical turns.

For example, the ends of the molecule in FIG. 15 could be joined to along DNA double helix. The ends of these long DNA segments could becovalently joined to make an interwound pair of circles with the DNAside of the switch region on one strand, and the RNA side on the other.If the circular DNAs are twisted in the proper superhelical direction,enough energy can be stored to cause a local cross-shaped, “cruciform”that extrudes the P and C loops as in the right side of FIG. 15. Thiscomes at a cost in free energy, ΔG° (cru). That is because base pairsare lost for the two loops at the ends of P and C, and there isadditional disruption at the branch of the cross. When ΔG° (sup) has alarge enough magnitude, the ΔG° (cru) penalty is overcome, and thecruciform extrudes. The point at which cruciform structures and doublehelix have equal populations in equilibrium is when ΔG°(cru)=ΔG° (sup).

This sets up a very similar situation to that described above, where theK1 equilibrium constant was adjusted by changing the nucleic acidsequence to set the trigger for a large change in luminescence (O→H) tooccur upon addition of a small amount of an effective competitor, C. Ifa ligand, like NCp7, is present in high enough concentration with thesuperhelical switch, it will bind the GGUG tetraloop and force thecruciform to occur at a lower density of superhelical turns. This can bequantified using free energies, which are additive; now the equalbalance between cruciform and double-helical structures occurs when ΔG°(cru)=ΔG° (sup)+ΔG(L/P). The balance between the two sides of thisequation can be adjusted so that addition of a small amount ofcompetitor ties up enough L to reduce the ΔG(L/P) contribution, andswitch the state away from the cruciform state. The adjustment in thiscase is accomplished by changing ΔG° (sup), which is a simple functionof the number of superhelical turns. Sequence-based tuning, asillustrated earlier, combined with balancing via superhelix density isattractive for the development of suitable bistable constructs. Currentprocedures for creating superhelices create a range of superhelixdensities and relaxed molecules; it may prove necessary to purifyconstructs within a relatively small range of superhelix densities.

Readout of the state of the cruciform/helix switch can again beaccomplished by fluorophore-quencher pairs. If these are positioned nearthe cruciform loops, the cruciform state will be highly luminescent andthe helix form dark. Positioning * and Q near the ends of the coverhairpin (analogous to FIG. 11) will produce the opposite result.

Molecular Electronic Applications

Certain molecular electronic applications can be realized using anembodiment of the present invention. This application also serves toillustrate methodology for integrating combinatorial sequence technologywith H to O switching. For example, FIG. 16 outlines the heart of aRead/Write/Erase device for information storage. The top panel indicatesthat the oligonucleotide-lumiphore/quencher “switch” can exist in a“zero” or a “one” conformation that can be toggled by light offrequencies, ν1 and ν2. The state of the switch can be interrogated bylight at ν3; the zero state has a very low luminescence emission at ν4,while the one state has robust emission. The device has only Read/Writecapability if the zero to one conversion is not capable of beingreversed by ν2. (The energy of a photon is represented by hνi, wherePlanck's constant is h. The wavelength of light is λi=c/νi, wherec=speed of light.)

The bottom panel of FIG. 16 illustrates an embodiment of the principlesjust discussed. An H-type molecule, similar to that in FIG. 11, is shownattached at its bottom to a solid support (S) via covalent attachment ofone of the two strands of the fastener duplex; such a solid-supportattachment may also be useful in some applications of the technologiesdescribed for diagnostics and screening presented previously. Inmolecular electronic applications this attachment provides spatialaddressability. The embodiment illustrated in FIG. 16 has a very similararrangement of lumiphore (*) and quencher (Q) to that illustrated inFIG. 11, and it can be seen that H to O conformational equilibria arestill common features. However, a difference lies in the attachment of aphotosensitive chemical entity to the 3′-end of the RNA/DNA chain by aflexible linker. The photochemical entity is illustrated in the figureas having two states, L1 which is converted to L2 by irradiation at ν1;if the erase function is to be implemented, L2 must be capable ofefficient back conversion by the action of ν2. When the photochemicalentity is in the L2 state, the O₂ to H2 equilibrium will favor H2, justas in the screening interaction illustrated previously, and intenseluminescence will occur due to the long distance between * and Q. Onlythe O-form of the construct has a binding pocket with high affinity forL1, but low affinity for L2. Thus, prior to irradiation at ν1, thebinding free energy of L1 for the binding pocket drives the equilibriumto favor the “dark” O1-form with very low luminescence emission.Although there are four states illustrated in FIG. 16, theconcentrations of the H1 and O2 states will usually be very small; theyare essentially intermediates in the pathway to converting between thestable O1 and H2 forms. The binding pockets for L1 and L2 can beoptimized in combinatorial experiments with a large variety ofsequences.

Molecules known as fulgides and fulgimides can exist in states where acentral ring is open or closed photochemically. The forms can be cycledmany times by the action of light at two different wavelengths (Wolak,M. A. et al. (2002) J. Photochem. Photobiol. A, 147:39-44). Many otherphotochemical entities have been characterized, as well (Willner, I.(1997) Acc. Chem. Res., 30:347-356). An o-nitrobenzyl photochemistry(Zhang, K. & Taylor, J.- S. (2001) Biochemistry, 40:153-159) isparticularly useful for Read/Write (no erase) devices. Here the actionof light at λ1=365 nm cleaves the L1 ligand from the 3′-end of a DNAmolecule. After cleavage, the effective concentration of L1 near thebinding pocket is reduced by a large fraction, rendering the switch inthe permanently “on” H-form.

Such devices are quite practical for sensitive Read/Write informationstorage applications. The domain size for luminescence detection andwriting using laser light sources is limited only by diffraction at thewavelength used. Usually many fewer photons will be required for reading(luminescence) than for writing or erasing (photochemical rearrangementof bonds). Therefore, it is unlikely that reading will cause asufficient amount of photochemistry to practically reverse the writingand erasing steps. A single addressable domain may contain moleculeswith different photochemical ligands, L1/L2. This can be used to providewavelength discrimination within a domain in a manner similar to currentplans for holographic data storage devices (Wise, K. J. (2002) TrendsBiotechnol. 20:387-394). Likewise, different lumiphores with distinctabsorption and emission spectra can provide multicolored detection.

Variations of Molecular Switch Morphology

Other arrangements of lumiphore, quencher, binding pocket, and molecularconformation are possible for switching devices within the scope of thepresent invention. It is also not a general requirement that the nucleicacid portion be chimeric for many applications involvingcombinatorially-derived sequence binding pockets.

While a number of preferred embodiments of the invention and variationsthereof have been described in detail, other modifications and methodsof use will be readily apparent to those of skill in the art.Accordingly, it should be understood that various applications,modifications and substitutions may be made of equivalents withoutdeparting from the spirit of the invention or the scope of the claims.

1. A method to generate a multichain nucleic acid switch adapted toswitch from a first conformation to a second conformation upon ligandbinding, said switch comprising: a probe strand P comprising a ligandbinding domain; a switching framework comprising a cover strand (C); atether comprising a base-pair stem that holds P and C together while theswitch changes between the first and second conformations, wherein P andC are on different nucleic acid chains; and a signaling apparatuscomprising a combination of signaling entities, said method comprisingthe following steps: (i) altering two nucleic acid sequences (a) and (b)to introduce mismatches between P and C; (ii) measuring the signal forthe two nucleic acid sequences (a) and (b), thereby determining anequilibrium constant, K₁ for the switch from the first conformation tothe second conformation for each of the two sequences (a) and (b); (iii)adjusting the equilibrium constant, K₁ between 0.1-0.01 based on K₁ forsequences (a) and (b); and (iv) generating the multichain nucleic acidswitch having the equilibrium constant, K₁ between 0.1-0.01.
 2. Themethod of claim 1, wherein the mismatches between P and C are introducedby altering the sequence of the C strand, and a free energy differencebetween the two conformations, is used to estimate K₁.
 3. The method ofclaim 1, wherein K₁ is set to favor conformation
 1. 4. The method ofclaim 1, wherein a value of K₁ is experimentally verified.
 5. The methodof claim 4, wherein a signal of a candidate switch is used to determinesaid candidate's value of K₁ by interpolation between that for sequence(a), which favors conformation 1 over conformation 2 by a factor of 100or more, and sequence (b), which favors conformation 2 over conformation1 by a factor of 100 or more.
 6. The method of claim 4, wherein thevalue of K₁ for switch candidates is estimated by comparison with knowncompetitors, X, of the ligand, L, designed to interact with conformation2 of the switch.
 7. A method to generate a branched, unimolecularnucleic acid switch adapted to switch from a first conformation to asecond conformation upon ligand binding, said switch comprising: a probestrand P comprising a ligand binding domain; a switching frameworkcomprising a cover strand (C); a toggle strand (T); a tether at a singlevertex that holds P, C and T together while the switch changes betweenthe first and second conformations; and a signaling apparatus comprisinga combination of signaling entities said method comprising the followingsteps: (i) altering sequences of C and/or T to introduce mismatchesbetween C and T in a sequence (a) which favors the first conformationand a sequence (b) which favors the second conformation; (ii) measuringthe signal for sequences (a) and (b), thereby determining an equilibriumconstant, K₁ for each of the two sequences (a) and (b); (iii) adjustingthe equilibrium constant, K₁, based upon the free energy differencebetween the K₁ for each of the two sequences (a) and (b); and (iv)generating the branched, unimolecular nucleic acid switch having theadjusted equilibrium constant, K₁.
 8. The method to generate amultichain nucleic acid switch according to claim 1, wherein the probestrand is selected from the group consisting of DNA, RNA, modifiednucleic acid, and combinations thereof.
 9. The method to generate amultichain nucleic acid switch according to claim 1, wherein thesignaling entities comprise a lumiphore and a quencher located along theswitching framework.
 10. The method to generate a multichain nucleicacid switch according to claim 1, wherein the ligand binding domaincomprises a naturally occurring RNA binding site or analog thereof or anaturally occurring DNA binding site or analog thereof or acombinatorially derived sequence or related fragment.
 11. The method togenerate a multichain nucleic acid switch according to claim 1, whereinthe ligand binding domain is adapted to bind a ligand which is selectedfrom the group consisting of: a disease agent, wherein the disease isHepatitis C, Congo-Crimean hemorrhagic fever, Ebola hemorrhagic fever,Herpes, human cytomegalovirus, human pappiloma virus, influenza,Marburg, Q fever, Rift valley fever, Smallpox, Venezuelan equineencephalitis, HIV-1, MMTV, HIV-2, HTLV-1, SNV, BIV, BLV, EIAV, FIV,MMPV, Mo-MLV, Mo-MSV, M-PMV, RSV, SIV, or AMV; a retroviral componentwhich is TAR-tat, RRE-rev, DIS, PBS, RT, PR, IN, SU, TM, vpu, vif, vpr,nef, mos, tax, rex, sag, v-src, v-myc and precursors and proteaseproducts of the precursors, gag, gag-pol, env, src, or onc; a toxin orother factor derived from bacteria or other microorganisms which are B.anthracis, Burkholderia pseudomallei, Botulinum, Brucellosis, Candidaalbicans, Cholera, Clostridium perfringins, Kinetoplasts, Malaria,Mycobacteria, Plague, Pneumocystis, Schistosomal parasites,Cryptosporidium, Giardia, Ricin, Saxitoxin, Shiga Toxin, Staphylococcus(including enterotoxin B), Trichothecene mycotoxins, Tularemia, oragents causing Toxoplasmosis; and nerve gas agents, chemical poisons,contaminants of water supplies, contaminants of food and beverages, orcontaminants of air.
 12. The method to generate a multichain nucleicacid switch according to claim 1, wherein at least some of themismatches between P and C in the first conformation are matched in thesecond conformation.
 13. The method to generate a multichain nucleicacid switch according to claim 1, wherein the tether is a fastenerduplex in two pieces.
 14. The method to generate a branched,unimolecular nucleic acid switch according to claim 7, wherein thesignaling apparatus comprises a lumiphore and a quencher of thelumiphore.
 15. The method to generate a branched, unimolecular nucleicacid switch according to claim 7, wherein the ligand binding domaincomprises RNA, the switching framework comprises DNA and the ligand is aviral protein.
 16. The method to generate a branched, unimolecularnucleic acid switch according to claim 7, wherein the ligand bindingdomain comprises RNA, the switching framework comprises DNA and theligand binding domain comprises a naturally occurring RNA binding siteor analog thereof or a naturally occurring DNA binding site or analogthereof or a combinatorially derived sequence or related fragment. 17.The method to generate a branched, unimolecular nucleic acid switchaccording to claim 7, wherein the ligand binding domain comprises RNA,the switching framework comprises DNA and the ligand is selected fromthe group consisting of: a disease agent wherein the disease isHepatitis C, Congo-Crimean hemorrhagic fever, Ebola hemorrhagic fever,Herpes, human cytomegalovirus, human pappiloma virus, influenza,Marburg, Q fever, Rift valley fever, Smallpox, Venezuelan equineencephalitis, HIV-1, MMTV, HIV-2, HTLV-1, SNV, BIV, BLV, EIAV, FIV,MMPV, Mo-MLV, Mo-MSV, M-PMV, RSV, SIV, or AMV; a retroviral componentwhich is TAR-tat, RRE-rev, DIS, PBS, RT, PR, IN, SU, TM, vpu, vif, vpr,nef, mos, tax, rex, sag, v-src, v-myc and precursors and proteaseproducts of the precursors, gag, gag-pol, env, src, or onc; a toxin orother factor derived from bacteria or other microorganisms which are B.anthracis, Burkholderia pseudomallei, Botulinum, Brucellosis, Candidaalbicans, Cholera, Clostridium perfringins, Kinetoplasts, Malaria,Mycobacteria, Plague, Pneumocystis, Schistosomal parasites,Cryptosporidium, Giardia, Ricin, Saxitoxin, Shiga Toxin, Staphylococcus(including enterotoxin B), Trichothecene mycotoxins, Tularemia, oragents causing Toxoplasmosis; and nerve gas agents, chemical poisons,contaminants of water supplies, contaminants of food and beverages, orcontaminants of air.
 18. The method to generate a branched, unimolecularnucleic acid switch according to claim 7, wherein the vertex comprises aphosphoramidite.